Increased fungal resistance in crop plants

ABSTRACT

The present invention relates to methods for producing plants with increased fungal resistance, preferably seedling resistance against Northern Corn Leaf Blight. Further provided are methods for introducing, modifying, or modulating at least one wall-associated kinase (WAK) in(to) a plant cell, tissue, organ, or whole plant and thereby causing a reduced synthesis of benzoxazinoid and in turn increased fungal resistance. There are further provided methods to identify and/or modify downstream effector molecules in a WAK signalling cascade. Finally, plant cells, tissues, organs or whole plants having increased fungal resistance and methods using substances to activate signalling pathways in a targeted way are provided. The present invention thus relates to WAKs as master regulators and crucial signaling mediators in plant defense against fungal disease and the regulation and cross-talk mechanisms in the WAK signaling cascade and further gives examples for establishing novel anti-fungal strategies relevant for a series of crop plants.

TECHNICAL FIELD

The present invention relates to methods for producing plants withincreased fungal resistance, preferably seedling resistance againstNorthern Corn Leaf Blight. Further provided are methods for introducing,modifying, or modulating at least one wall-associated kinase (WAK)in(to) a plant cell, tissue, organ, or whole plant and thereby causing areduced synthesis of benzoxazinoid and in turn increased fungalresistance. There are further provided methods to identify and/or modifydownstream effector molecules in a WAK signalling cascade. Finally,plant cells, tissues, organs or whole plants having increased fungalresistance and methods using substances to activate signalling pathwaysin a targeted way are provided. The present invention thus relates toWAKs as master regulators and crucial signaling mediators in plantdefense against fungal disease and the regulation and cross-talkmechanisms in the WAK signaling cascade and further gives examples forestablishing novel anti-fungal strategies relevant for a series of cropplants.

BACKGROUND OF THE INVENTION

Infections and infestations of crop plants by pathogens encompassingviruses, bacteria, fungi, nematodes and insects and the resultingdamages cause significant yield losses of cultivated plants. In maize(Zea mays) as one of the major crop plants worldwide there are a largenumber of fungal pathogens which cause leaf diseases. The fungus whichcan cause by far the most damage under tropical and also under temperateclimatic conditions, such as those in large parts of Europe and NorthAmerica as well as in Africa and India, is known as Helminthosporiumturcicum or synonymously as Exserohilum turcicum (teleomorph:Setosphaeria turcica). H. turcicum/E. turcicum is the cause of the leafspot disease known as “Northern Corn Leaf Blight” (NCLB), which canoccur in epidemic proportions during wet years, attacking vulnerablemaize varieties and causing a great deal of damage and considerablelosses of yield of 30% and more over wide areas (Perkins, J. M., and W.L. Pedersen. “Disease development and yield losses associated withnorthern leaf blight on corn.” Plant disease 71.10 (1987): 940-943;Raymundo, A. D., A. L. Hooker, and J. M. Perkins. “Effect of gene HtN onthe development of northern corn leaf blight epidemics.” Plant disease(1981); Ullstrup, A J, and Miles, S R 1957. The effects of some leafblights of corn on grain yield. Phytopathology 47:331-336). Since the1970s, then, natural resistance in genetic material has been sought.

The race for defining and establishing new resistance strategies againstpathogens for major crop plants is more and more accelerated due to theincreasing resistance breaking characteristics of pathogens, i.e., theevolutionary strategy of pathogens to adapt to and survive pressure ofplant protective agents and/or to subvert the endogenous plant defensemechanisms.

Currently, quantitative and qualitative resistances are known. While theoligo- or polygenically inherited quantitative resistance appearsincomplete and non-specific as regards race in the phenotype and isinfluenced by additional and partially dominant genes, qualitativeresistance is typically race-specific and can be inherited throughindividual: For instances, mostly dominant resistance genes with regardto NCLB are Ht1, Ht2, Ht3, Htm, Htn1, or Htp (Lipps, P. E., R. C. Pratt,and J. J. Hakiza. “Interaction of Ht and partial resistance toExserohilum turcicum in maize.” Plant disease 81.3 (1997): 277-282;Wang, H., et al. “Expression of Ht2-related genes in response to theHT-Toxin of Exserohilum turcicum in Maize.” Annals of applied biology156.1 (2010): 111-120). Backcrosses in many frequently used inbred maizelines such as W22, A619, B37 or B73 have successfully brought aboutintrogression of the HT genes, where they exhibit a partial dominanceand expression as a function of the respective genetic background (Welz,H G (1998). “Genetics and epidemiology of the pathosystem Zeamays/Setosphaeria turcica.” Habilitationsschrift Institut fürPflanzenzüchtung, Saatgutforschung and Populationsgenetik, UniversitätHohenheim). WO 2011/163590 A1 annotated the presumed Htn1 gene in theresistance source PH26N and PH99N as a tandem protein kinase-like geneand disclosed its genetic sequence, but did not determine itsfunctionality, for example in a transgenic maize plant. WO 2015/032494A2 discloses the identification of another allelic variant of HTN1 genederived from the donor Peptilla as well as a resistant maize plant intothe genome of which a chromosome fragment from the donor Pepitilla hasbeen integrated, which chromosome fragment comprises the resistancelocus HTN1.

The hemibiotrophic fungal pathogen Exserohilum turcicum (anamorph formof the fungus) causing NCLB is found in humid climates wherever corn isgrown. E. turcicum survives in corn debris and builds up over time inhigh-residue and continuous corn cropping systems. High humidity andmoderate temperatures favor the persistence of the E. turcicum funguscausing tremendous yield losses, e.g., due to decreased photosynthesisresulting in limited ear fill, or harvest losses if secondary stalk rotinfection and stalk lodging accompany loss of leaf area.

As a natural defense mechanism against various kinds of pathogens,Plants have evolved multiple layers of defense against infection bypathogenic microbes (Jones, Jonathan DG, and Jeffery L. Dangl. “Theplant immune system.” Nature 444.7117 (2006): 323). The primary defenseis based on the extracellular perception of pathogen-associated or hostdamage-associated molecular patterns or signatures (PAMPs/DAMPs) byplasma membrane-anchored pattern recognition receptors (PRRs). Thesereceptor proteins monitor the extracellular space for the presence ofmicrobial- or host-derived elicitors, i.e., PAMPs or DAMPs,respectively. These signatures can be highly conserved andcharacteristic for entire pathogen classes as in the case of thebacterial flagellin that is perceived by the leucine-rich repeatreceptor kinase (LRR-RK) FLS2, which results in basal and broad-spectrumresistance against most bacteria (Macho, Alberto P., and Cyril Zipfel.“Plant PRRs and the activation of innate immune signaling.” Molecularcell 54.2 (2014): 263-272). Other receptor kinases only conferresistance to certain races of a particular pathogen (Hu, Keming, et al.“Improvement of multiple agronomic traits by a disease resistance genevia cell wall reinforcement.” Nature plants 3 (2017): 17009). Receptorkinases have different types of extracellular domains, includingleucine-rich repeats (LRRs), lysine motifs (LysMs), lectin motifs orepidermal growth factor (EGF) like extracellular domains (Dardick,Chris, Benjamin Schwessinger, and Pamela Ronald. “Non-arginine-aspartate(non-RD) kinases are associated with innate immune receptors thatrecognize conserved microbial signatures.” Current opinion in plantbiology 15.4 (2012): 358-366).

In grasses, there is emerging evidence that WAKs might be importantplayers in fungal and bacterial disease resistance. The WAK genes qHSR1,Htn1 and OsWAK (Xa4) confer disease resistance (Hurni, Severine, et al.“The maize disease resistance gene Htn1 against northern corn leafblight encodes a wall-associated receptor-like kinase.” Proceedings ofthe National Academy of Sciences 112.28 (2015): 8780-8785., Hu et al.2017), yet little is known about the underlying mechanisms and signalingcascades responsible for the observed phenotypes.

OsWAK underlying resistance involves strengthening of the cell intensityby enhancing cellulose biosynthesis (Hu et al. 2017). Interestingly,there is also one case described where the wheat WAK encoded by the Snn1gene acts as a susceptibility factor. It has been shown that Snn1perceives the SnTox1 toxin encoded by the fungal pathogen Stagonosporanodorum, which triggers cell death and allows the necrotrophic S.nodorum pathogen to proliferate on wheat (Shi, Gongjun, et al. “Markerdevelopment, saturation mapping, and high-resolution mapping of theSeptoria nodorum blotch susceptibility gene Snn3-B1 in wheat.” Moleculargenetics and genomics 291.1 (2016): 107-119). In dicots, the ArabidopsisAtWAK1 was found to physically associate with and recognized cellwall-derived oligogalactouronides (OGs), which result frompolysaccharide degradation (Kohorn, Bruce D., et al. “Pectin activationof MAP kinase and gene expression is WAK2 dependent.” The Plant Journal60.6 (2009): 974-982). In contrast to the Arabidopsis WAK gene familythat consists of only five members, WAK genes in monocots belong to alarge family. For instance, in rice >100 members were found (Kanneganti,Vydehi, and Aditya K. Gupta. “Wall associated kinases from plants anoverview.” Physiology and Molecular Biology of Plants 14.1-2 (2008):109-118). The emergence of WAKs in monocots might be associated toseveral functional aspects, e.g. biotic diseases (Li, Hui, et al. “Anovel wall-associated receptor-like protein kinase gene, OsWAK1, playsimportant roles in rice blast disease resistance.” Plant molecularbiology 69.3 (2009): 337-346; Hurni et al. 2015; Zuo, Weiliang, et al.“A maize wall-associated kinase confers quantitative resistance to headsmut.” Nature genetics 47.2 (2015): 151-157; Shi, Gongjun, et al. “Thehijacking of a receptor kinase-driven pathway by a wheat fungal pathogenleads to disease.” Science advances 2.10 (2016): e1600822; Hu et al.2017), tolerance of phosphorus deficiency (Hufnagel, Barbara, et al.“Duplicate and conquer: Multiple homologs of PHOSPHORUS-STARVATIONTOLERANCE1 enhance phosphorus acquisition and sorghum performance onlow-phosphorus soils.” Plant physiology 166.2 (2014): 659-677), rootgrowth (Kaur, Ravneet, Kashmir Singh, and Jaswinder Singh. “Aroot-specific wall-associated kinase gene, HvWAK1, regulates root growthand is highly divergent in barley and other cereals.” Functional &integrative genomics 13.2 (2013): 167-177) as well as gametophytedevelopment (Wang, Na, et al. “The rice wall-associated receptor-likekinase gene OsDEES1 plays a role in female gametophyte development.”Plant physiology 160.2 (2012): 696-707; Hu et al. 2017). Therefore,there is emerging evidence that wall-associated kinases (WAKs) couldplay a pivotal role in plant immunity specifically in cereal crops.

Still, presently little is known about the specific molecular mechanismsunderlying plant immunity based on WAKs, said immune mechanisms beinghighly specific for each pathogen and thus PAMP/DAMP to be recognizedand furthermore depending on the downstream signaling cascade and thesubsequently initiated effector mechanisms. So far, rather thephenotypic outcome of a plant comprising or not comprising areceptor-like kinase on pathogen resistance has been matched with thegenotype of a plant. The molecular mechanisms which are responsible forthe action of the WAKs, the precise signaling pathways and also thecrosstalk between different pathways remain elusive.

Chemical fungicides have long been utilised for controlling fungaldiseases. A different approach relies on the examination and elucidationof the complex biosynthetic pathways involved in pathogen resistancecausing a natural pathogen defence of plants by studying the resistanceability of an existing plant cultivar to inhibit or at least limit anyinfestation of a pathogen to provide new strategies to combat plantpathogens and to provide new plants carrying resistance traits ofinterest.

Benzoxazinoids (BXDs) were identified in the 1960s as secondary plantmetabolites functioning as natural pesticides. BXDs are a class ofsecondary metabolites found in maize and other cereal species andcertain dicots and contain a 2-hydroxy-2H-1,4-benzoxazin-3(4H)-oneskeleton (Niemeyer, Hermann M. “Hydroxamic acids derived from2-hydroxy-2 H-1, 4-benzoxazin-3 (4 H)-one: key defense chemicals ofcereals.” Journal of Agricultural and Food Chemistry 57.5 (2009):1677-1696). BXDs are synthesized in seedlings and stored as glucosides.The main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one(DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one(DIMBOA). BXDs are synthesised in two subfamilies of the Poaceae andsporadically found in single species of the dicots. BXDs arepredominantly stored as inactive glucosides, while upon biotic stressthey are hydrolyzed to the respective toxic hydroxamic acids (e.g.,DIMBOA). The first step in BXD biosynthesis converts indole-3-glycerolphosphate into indole. In maize (Zea mays), this reaction is catalyzedby either Benzoxazineless1 (BX1), or Indole glycerol phosphate lyase(IGL or Igl herein). The bx1 gene is under developmental control and ismainly responsible for BX production, whereas the Igl gene is inducibleby stress signals, such as wounding, herbivory, or jasmonates. Theenzymatic properties of IGL are similar to BX1, but the transcriptionalregulation of their corresponding genes is different. Like other Bxgenes, bx1 is constitutively expressed during the early developmentalstages of the plant, which correlates with endogenous BX levels. Theintroduction of four oxygen atoms into the indole moiety that yieldsDIBOA is catalyzed by four cytochrome P450 monooxygenases termed BX2 toBX5. A further Bx enzyme, BX6, is responsible for the hydroxylation inposition C-7 of the benzoxazinoids in maize (Frey, Monika, et al. “A2-oxoglutarate-dependent dioxygenase is integrated inDIMBOA-biosynthesis.” Phytochemistry 62.3 (2003): 371-376). Bx7, asfurther example, is an O-methyltransferase (OMT) catalyzing theformation of DIMBOA-glc from TRIBOA-glc.

Little is known about the role of these compounds in fungal diseaseresistance. Few field studies decades ago proposed a correlation betweenbenzoxazinoid hydroxamic acids concentration and the disease resistanceto maize stalk rot, maize northern corn leaf blight and wheat stem rust(Long, B. J., G. M. Dunn, and D. G. Routley. “Relationship ofhydroxamate concentration in maize and field reaction toHelminthosporium turcicum.” Crop Science 18.4 (1978): 573-575) withoutproviding any molecular basis for this phenomenon. Other studies evenfound no effect of BXDs in fungal disease resistance, including maizestalk rot, Southern corn leaf blight, maize anthracnose, corn smut andhead blight at all (Niemeyer, 2009).

Therefore, it was an outstanding aim of the present invention to providenew tools and methods to allow for a tight resistance management formajor crop plants concerning the control of fungal plant pathogens.

Further, it was an object of the present invention to provide newstrategies and methods to combat fungal leaf diseases based onexploiting plant-endogenous defense mechanisms as mediated by secondarymetabolites and further to provide plants having a specific genotype assource of increased resistance or tolerance against fungal pathogenscausing severe plant diseases. Basically, it was an object of thepresent invention to elucidate the molecular basis of the maize Htn1northern corn leaf blight (NCLB) resistance that is cause by the WAKgene ZmWAK-RLK1 to establish molecular targets and cross-talk mechanismsbetween relevant biosynthesis pathways to provide new urgently neededresistance strategies for a variety of important crop plants bycharacterizing the molecular players involved in disease resistance in aplant.

SUMMARY OF THE INVENTION

The above object was achieved by identifying the molecular basis of themaize Htn1 northern corn leaf blight (NCLB) resistance that is caused bythe WAK gene ZmWAK-RLK1. It was demonstrated that ZmWAK-RLK1 modulates,i.e. it functions upstream of the benzoxazinoids (BXD) biosynthesispathway, resulting in reduced BXD concentrations. Furthermore, theinteraction of WAK with downstream effectors, i.e., BX enzymes and Igl,in the regulation of BXD biosynthesis and fungal disease weredemonstrated. Furthermore, the present invention builds on relevantinformation of the cross-talk and cross-regulation of the plant WAKsignaling pathway with the jasmonic acid (JA) and further relevant plantbiosynthesis pathways to elucidate the regulatory networks important tomodulate and to induce plant defense against major pathogens.

Interestingly, maize plants with compromised BXD biosynthesis showedincreased resistance against NCLB. Thus, these new insight intoWAK-mediated quantitative disease resistance and for the first timeprovided a functional link between WAKs and the secondary metabolitesBXDs which was used to define new fungal defense strategies going beyondthe use of fungicides for disease control which are applicable forseveral associated fungal pathogens inducing comparable plant immunereactions by pathogen-associated PAMPs/DAMPs in several important cropplants.

The present invention thus transforms the information on molecularmechanisms, the effect of specific mutations in kinase domains on thesignaling function of a WAK, and further on the cross-talk and interplayof signaling cascades to provide plants having a defined geneticbackground and thus to provide plants with increased resistance againstfungal pathogens. Furthermore, the present invention provides methods tomodulate (increase/decrease) or neutralize the action of plant secondarymetabolites and the relevant genes responsible for the synthesispathways of said secondary metabolites to enhance fungal pathogenresistance in a targeted way.

In one aspect, there is thus provided a method for producing a planthaving increased fungal resistance as compared to a correspondingcontrol plant, wherein the fungal resistance is regulated by at leastone wall-associated kinase, the method comprising: (i) (a) providing atleast one plant cell, tissue, organ, or whole plant having a specificgenotype with respect to the presence of at least one gene encoding awall-associated kinase in the genome of said plant cell, tissue, organ,or whole plant; or (i) (b) introducing at least one gene encoding atleast one wall-associated kinase into the genome of at least one cell ofat least one of a plant cell, tissue, organ, or whole plant; and (ii)(a) modifying at least one gene encoding at least one wall-associatedkinase in the at least one plant cell, tissue, organ, or whole plant;and/or (ii) (b) modulating the expression level of at least onewall-associated kinase and/or the transcription level, the expressionlevel, or the function of at least one molecule within the signallingpathway from the at least one wall-associated kinase to the synthesis ofat least one benzoxazinoid or within the synthesis pathway of at leastone benzoxazinoid in the at least one plant cell, tissue, organ, orwhole plant; (iii) producing a population of plants from the at leastone plant cell, tissue, organ, or whole plant, and (iv)selecting/identifying a plant having increased fungal resistance, fromthe population based on the determination of a reduced synthesis of atleast one benzoxazinoid preferably in response to a fungal pathogeninfection, wherein the selected plant have an increased fungalresistance based on the reduced synthesis of a benzoxazinoid, and/orwherein the synthesis of the at least one benzoxazinoid is regulated bythe at least one wall-associated kinase.

In one embodiment of the various aspects of the present invention, theat least one wall-associated kinase is a WAK-RLK1 gene, preferablyselected from Htn1, Ht2, or Ht3, or an allelic variant thereof, a mutantor a functional fragment thereof, or a gene encoding the same,preferably wherein the at least one wall-associated kinase a) is encodedby a nucleic acid molecule comprising the nucleotide sequence of SEQ IDNO: 1 or 7, or a functional fragment thereof, b) is encoded by a nucleicacid molecule comprising the nucleotide sequence having at least 60%,65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity tonucleotide sequence of SEQ ID NO: 1 or 7, preferably over the entirelength of the sequence, c) is encoded a nucleic acid moleculehybridizing with a complementary sequence to a) or b) under stringentconditions, d) is encoded by a nucleic acid molecule comprising thenucleotide sequence coding for an amino acid sequence of SEQ ID NO: 2 or8, or a functional fragment thereof, e) is encoded by a nucleic acidmolecule comprising the nucleotide sequence coding for an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8,preferably over the entire length of the sequence, f) comprising theamino acid sequence of SEQ ID NO: 2 or 8, or g) comprising an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8,preferably over the entire length of the sequence, provided that the anysequence of a) to g), optionally after expression, still encodes atleast one functional Htn1, Ht2, or Ht3, or an allelic variant, a mutant,or a functional fragment thereof. Preferably, the at least onewall-associated kinase of the invention causes after expression areduced synthesis of at least one benzoxazinoid. More preferably, in Zeamays the gene or nucleic acid molecule encoding the at least onewall-associated kinase is located at or mapped on a locus in bin 5 orbin 6 on the long arm of chromosome 8.

In a further embodiment of the various aspects of the present invention,the benzoxazinoid whose biosynthesis is regulated by the at least onewall-associated kinase, is selected from at least one of DIM₂BOA,DIMBOA, HMBOA, HM₂BOA, HDMBOA, HDM₂BOA, HBOA, DHBOA, DIBOA or TRIBOA,the aforementioned benzoxazinoid being in the glucoside or agluconeform, or a benzoxazolinone, or any combination of the aforementionedbenzoxazinoids, preferably wherein the benzoxazinoid whose biosynthesisis regulated by the at least one wall-associated kinase is selected fromat least one of DIM₂BOA, DIMBOA, HMBOA or HDMBOA, the aforementionedbenzoxazinoid being in the glucoside or aglucone form, or anycombination of the aforementioned benzoxazinoids.

In another embodiment, there is provided a method, wherein the reducedsynthesis of at least one benzoxazinoid is achieved by providing atleast one wall-associated kinase, an allelic variant, a mutant or afunctional fragment thereof, or a gene encoding the same, wherein the atleast one wall-associated kinase comprises a sequence which can directlyor indirectly influence the benzoxazinoid (synthesis) pathway and atleast one further plant metabolic pathway, preferably a diseaseresistance associated pathway, wherein the plant metabolic pathway isselected from the group consisting of the jasmonic acid pathway, theethylene pathway, the lignin synthesis pathway, a defense pathway, areceptor-like kinase pathway, and/or a cell wall associated pathway.

In yet a further embodiment of the various aspects of the presentinvention, the fungus resistance against which resistance is increased,or the disease caused by said fungus is selected from a fungus of theorder of Pleosporales, comprising E. turcicum/H. turcicum causingnorthern corn leaf blight (NCLB), particularly affecting maize and wheatplants, southern corn leaf blight (Bipolaris maydis), the order ofPucciniales causing rust disease, comprising common rust (Pucciniasorghi), or Diploida leaf streak/blight (Diploidamacrospora/Stenocarpella macrospora), or Colletotrichum graminicola, orFusarium spp., preferably Fusarium verticilioides causing Fusarium stalkrot, or Gibberella spp., e.g., Gibberella zeae causing Giberella stalkrot, rust, stalk rot, maize head smut (Sphacelotheca reiliana), andDiploida leaf streak/blight.

In one of the embodiments of the various aspects of the presentinvention, the at least one gene encoding at least one wall-associatedkinase is stably integrated/introduced into the genome of the at leastone plant cell, tissue, organ, or whole plant, or the at least one geneencoding at least one wall-associated kinase is transiently introducedinto a plant cell, tissue, organ, or whole plant.

In a further embodiment of the various aspects of the present invention,the at least one molecule within the signalling pathway from the atleast one wall-associated kinase to the synthesis of at least onebenzoxazinoid or within the synthesis pathway of at least onebenzoxazinoid is selected from the group consisting of the genes bx1(SEQ ID NO: 10), bx2 (SEQ ID NO: 12), igl (SEQ ID NO: 14), bx6 (SEQ IDNO: 16), bx11 (SEQ ID NO: 18), bx14 (SEQ ID NO: 20), opr2 (SEQ ID NO:22), lox3 (SEQ ID NO: 24) or aoc1 (SEQ ID NO: 26), or a homologous genesthereof, or the proteins BX1 (SEQ ID NO: 11), BX2 (SEQ ID NO: 13), IGL(SEQ ID NO: 15), BX6 (SEQ ID NO: 17), BX11 (SEQ ID NO: 19), BX14 (SEQ IDNO: 21), OPR2 (SEQ ID NO: 23), LOX3 (SEQ ID NO: 25) or AOC1 gene (SEQ IDNO: 27), or a homolog thereof.

In another embodiment of the various aspects of the present invention,the at least one gene encoding at least one wall-associated kinase isstably integrated into the genome of the at least one plant cell,tissue, organ, or whole plant, and the introduction of the at least onegene encoding at least one wall-associated kinase comprises theintrogression of the at least one gene during plant breeding.

In a further embodiment of the of the various aspects of the presentinvention, the modification of the at least one gene encoding at leastone wall-associated kinase within step (ii) (a) or (ii) (b) of themethod of the above disclosed aspect is performed by at least one of asite-specific nuclease (SSN) or a catalytically active fragment thereof,or a nucleic acid sequence encoding the same, oligonucleotide directedmutagenesis, chemical mutagenesis, or TILLING.

In a further embodiment of the of the various aspects of the presentinvention, the at least one site-specific nuclease (SSN), or the nucleicacid sequence encoding the same, is selected from at least one of aCRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, ameganuclease, a base editor complex, a restriction endonuclease,including FokI or a variant thereof, or two site-specific nickingendonucleases, or a variant or a catalytically active fragment thereof.

According to one embodiment of the various aspects of the presentinvention, the at least one plant cell, tissue, organ, or whole plantprovided in step (i) is selected from the group consisting of Hordeumvulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zeaspp., including Zea mays, Setaria italica, Oryza minuta, Oryza sativa,Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum,Secale cereale, Triticale, Malus domestica, Brachypodium distachyon,Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta spp.,including Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucuscarota, Eucalyptus grandis, Nicotiana sylvestris, Nicotianatomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanumlycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera,Erythrante guttata, Genlisea aurea, Cucumis sativus, Marus notabilis,Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsis thaliana,Crucihimalaya himalaica, Crucihimalaya wallichii, Cardamine nexuosa,Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsis pumila,Arabis hirsute, Brassica napus, Brassica oleracea, Brassica rapa,Raphanus sativus, Brassica juncacea, Brassica nigra, Eruca vesicariasubsp. sativa, Citrus sinensis, Jatropha curcas, Populus trichocarpa,Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicer arietinum,Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius, Cajanusscarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp.,Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa,Allium fistulosum, Allium sativum, Helianthus annuus, Helianthustuberosus and Allium tuberosum, or any variety or subspecies belongingto one of the aforementioned plants, preferably wherein the plant cell,tissue, organ, or whole plant in step (i) is selected from Zea mays orTriticum spp., or any variety or subspecies belonging to one of theaforementioned plants

In a further aspect, there is provided a plant cell, tissue, organ,whole plant or plant material, or a derivative or a progeny thereof,obtainable by a method according to any one of the embodiments of themethods of the present invention.

In a further aspect of the present invention, there is provided a methodfor identifying at least one gene involved in increased pathogenresistance, preferably increased fungal resistance, in a plant cell,tissue, organ, whole plant, or plant material as compared to acorresponding control plant cell, tissue, organ, whole plant, or plantmaterial the method comprising: (i) determining the genotype of at leastone plant cell, tissue, organ, whole plant, or plant material withrespect to the presence of at least one gene encoding a wall-associatedkinase in the genome of said plant cell, tissue, organ, whole plant orplant material; (ii) optionally: determining the benzoxazinoid signatureof the at least one plant cell, tissue, organ, whole plant, or plantmaterial of step (i); (iii) exposing the at least one plant cell,tissue, organ, whole plant, or plant material of step (i) or (ii) to astimulus, optionally wherein the stimulus is correlated with thebenzoxazinoid signature in the at least one plant cell, tissue, organ,whole plant, or plant material, preferably wherein the stimulus isassociated with a fungal pathogen infection; (iv) performing an analysisof at least one analyte obtained from the at least one plant cell,tissue, organ, whole plant, or plant material of step (i) or (ii) afterexposition to the stimulus; (v) determining at least one gene beingregulated upon exposition to a stimulus according to step (iii) in atleast one cell of the at least one plant cell, tissue, organ, wholeplant, or plant material as derivable from the analysis of at least oneanalyte as defined in step (iv), (vi) subjecting the at least one geneas determined in step (v) to a functional characterization; and (vii)providing at least one gene involved in increased pathogen resistance,preferably increased fungal resistance, in a plant cell, tissue, organ,whole plant, or plant material.

Further provided is a plant cell, tissue, organ, whole plant or plantmaterial, or a derivative or a progeny thereof, obtainable byintroducing at least one gene as provided by the method for identifyingat least one gene involved in increased pathogen resistance into atleast one cell of at least one of a plant cell, tissue, organ, or wholeplant.

In another embodiment of the present invention, there is provided aplant cell, tissue, organ, whole plant or plant material, or aderivative or a progeny thereof, obtainable by the methods of thepresent invention, wherein the introduction of at least one gene asprovided by the method for identifying at least one gene involved inincreased pathogen resistance is a stable introduction, preferably astable introduction mediated by conventional plant breeding, or a stableintroduction mediated by means of molecular biology, comprising genomeediting, or a combination thereof.

In yet a further aspect of the present invention there is provided amethod of increasing pathogen resistance, preferably fungal resistance,in a plant cell, tissue, organ, whole plant, or plant material ascompared to a corresponding control plant cell, tissue, organ, wholeplant, or plant material, the method comprising: (i) providing at leastone plant cell, tissue, organ, whole plant or plant material; (ii) (a)treating the at least one plant cell, tissue, organ, whole plant orplant material according to step (i) with a substance neutralizing theeffect of at least one benzoxazinoid, and/or (ii) (b) treating the atleast one plant cell, tissue, organ, whole plant or plant materialaccording to step (i) with a substance activating the signalling pathwaydownstream of at least one wall-associated kinase; and/or (ii) (c)modifying at least one promoter or at least one regulatory sequence ofat least one gene of the at least one plant cell, tissue, organ, wholeplant or plant material of step (i), wherein said at least promoter orat least one regulatory sequence is involved in the regulation oftranscription of at least on gene involved in the signalling pathway of,or downstream of at least one wall-associated kinase; (iii) reducing theamount of at least one benzoxazinoid and thereby increasing pathogenresistance, preferably fungal resistance, in at least one plant cell,tissue, organ, whole plant, or plant material.

Further provided is a use of a substance as defined for the aspect of amethod of increasing pathogen resistance for increasing pathogenresistance, preferably fungal resistance, in at least one plant cell,tissue, organ, whole plant, or plant material.

Definitions

The term “active fragment” or “functional fragment” as used hereinreferring to amino acid sequences denotes the core sequence derived froma given template amino acid sequence, or a nucleic acid sequenceencoding the same, comprising all or part of the active site of thetemplate sequence with the proviso that the resulting catalyticallyactive fragment still possesses the activity characterizing the templatesequence, for which the active site of the native enzyme or a variantthereof is responsible. Said modifications are suitable to generate lessbulky amino acid sequences still having the same activity as a templatesequence making the catalytically active fragment a more versatile ormore stable tool being sterically less demanding. For amino acidsequences not representing enzymes, the term “functional fragment” canalso imply that part or domain of the amino acid sequence involved ininteraction with another molecule, and/or involved in any structuralfunction within the cell.

An “allele” or “allelic variant” as used herein refers to a variant formof a given gene. As most multicellular organisms have two sets ofchromosomes; that is, they are diploid (or, if more chromosome sets arepresent, they are polyploidy), these chromosomes are referred to ashomologous chromosomes. If both alleles at a gene (or locus) on thehomologous chromosomes are the same, they and the organism arehomozygous with respect to that gene (or locus). If the alleles aredifferent, they and the organism are heterozygous with respect to thatgene. Alleles can result in the same, or a different observablephenotype. The term “allele” thus refers to one or two or morenucleotide sequences at a specific locus in the genome. A first alleleis on a chromosome, a second on a second chromosome at the sameposition. If the two alleles are different, they are heterozygous, andif they are the same, they are homozygous. Various alleles of a gene(gene alleles) differ in at least one SNP (single nucleotidepolymorphism).

“Complementary” or “complementarity” as used herein describes therelationship between two DNA, two RNA, or, regarding hybrid sequencesaccording to the present invention, between an RNA and a DNA nucleicacid region. Defined by the nucleobases of the DNA or RNA, two nucleicacid regions can hybridize to each other in accordance with thelock-and-key model. To this end the principles of Watson-Crick basepairing have the basis adenine and thymine/uracil as well as guanine andcytosine, respectively, as complementary bases apply. Furthermore, alsonon-Watson-Crick pairing, like reverse-Watson-Crick, Hoogsteen,reverse-Hoogsteen and Wobble pairing are comprised by the term“complementary” as used herein as long as the respective base pairs canbuild hydrogen bonding to each other, i.e., two different nucleic acidstrands can hybridize to each other based on said complementarity.

The term “construct”, especially “genetic construct”, or “recombinantconstruct”, or “expression construct” as used herein refers to aconstruct comprising, inter alia, plasmids or plasmid vectors, cosmids,artificial yeast chromosomes or bacterial artificial chromosomes (YACsand BACs), phagemides, bacterial phage based vectors, an expressioncassette, isolated single-stranded or double-stranded nucleic acidsequences, comprising DNA and RNA sequences, or amino acid sequences,viral vectors, including modified viruses, and a combination or amixture thereof, for introduction or transformation, transfection ortransduction into a target cell or plant, plant cell, tissue, organ ormaterial according to the present disclosure.

The term “delivery construct” or “delivery vector” as used herein refersto any biological or chemical means used as a cargo for transporting anucleic acid, including a hybrid nucleic acid comprising RNA and DNA,and/or an amino acid sequence of interest into a target cell, preferablya eukaryotic cell. The term delivery construct or vector as used hereinthus refers to a means of transport to deliver a genetic or arecombinant construct according to the present disclosure into a targetcell, tissue, organ or an organism. A vector can thus comprise nucleicacid sequences, optionally comprising sequences like regulatorysequences or localization sequences for delivery, either directly orindirectly, into a target cell of interest or into a plant targetstructure in the desired cellular compartment of a plant. A vector canalso be used to introduce an amino acid sequence or aribonucleo-molecular complex into a target cell or target structure.Usually, a vector as used herein can be a plasmid vector. Furthermore,according to certain preferred embodiments according to the presentinvention, a direct introduction of a construct or sequence or complexof interest is conducted. The term direct introduction implies that thedesired target cell or target structure containing a DNA target sequenceto be modified according to the present disclosure is directlytransformed or transduced or transfected into the specific target cellof interest, where the material delivered with the delivery vector willexert its effect. The term indirect introduction implies that theintroduction is achieved into a structure, for example, cells of leavesor cells of organs or tissues, which do not themselves represent theactual target cell or structure of interest to be transformed, but thosestructures serve as basis for the systemic spread and transfer of thevector, preferably comprising a genetic construct according to thepresent disclosure to the actual target structure, for example, ameristematic cell or tissue, or a stem cell or tissue. In case the termvector is used in the context of transfecting amino acid sequencesand/or nucleic sequences, including hybrid nucleic acid sequences, intoa target cell the term vector implies suitable agents for peptide orprotein transfection, like for example ionic lipid mixtures, cellpenetrating peptides (CPPs), or particle bombardment. In the context ofthe introduction of nucleic acid material, the term vector cannot onlyimply plasmid vectors but also suitable carrier materials which canserve as basis for the introduction of nucleic acid and/or amino acidsequence delivery into a target cell of interest, for example by meansof particle bombardment. Said carrier material comprises, inter alia,gold or tungsten particles. Finally, the term vector also implies theuse of viral vectors for the introduction of at least one geneticconstruct according to the present disclosure like, for example,modified viruses and bacterial vectors, like for example Agrobacteriumspp., like for example Agrobacterium tumefaciens. Finally, the termvector also implies suitable chemical transport agents for introducinglinear nucleic acid sequences (single- or double-stranded), or aminosequences, or a combination thereof into a target cell combined with aphysical introduction method, including polymeric or lipid-baseddelivery constructs.

Suitable “delivery constructs” or “vectors” thus comprise biologicalmeans for delivering nucleotide and/or amino acid sequences into atarget cell, including viral vectors, Agrobacterium spp., or chemicaldelivery constructs, including nanoparticles, e.g., mesoporous silicananoparticles (MSNPs), cationic polymers, including PEI(polyethylenimine) polymer based approaches or polymers likeDEAE-dextran, or non-covalent surface attachment of PEI to generatecationic surfaces, lipid or polymeric vesicles, or combinations thereof.Lipid or polymeric vesicles may be selected, for example, from lipids,liposomes, lipid encapsulation systems, nanoparticles, small nucleicacid-lipid particle formulations, polymers, and polymersomes.

The term “derivative” or “descendant” or “progeny” as used herein in thecontext of a prokaryotic or a eukaryotic cell, preferably a plant orplant cell or plant material according to the present disclosure relatesto the descendants of such a cell or material which result from naturalreproductive propagation including sexual and asexual propagation. It iswell known to the person having skill in the art that said propagationcan lead to the introduction of mutations into the genome of an organismresulting from natural phenomena which results in a descendant orprogeny, which is genomically different to the parental organism orcell, however, still belongs to the same genus/species and possessesmostly the same characteristics as the parental recombinant host cell.Such derivatives or descendants or progeny resulting from naturalphenomena during reproduction or regeneration are thus comprised by theterm of the present disclosure. These terms, therefore, do not refer toany arbitrary derivative, descendant or progeny, but rather to aderivative, or descendant or progeny phylogenetically associated with,i.e., based on, a parent cell thereof, whereas this relationship betweenthe derivative, descendant or progeny and the “parent” is clearlyinferable by a person skilled in the art. “Progeny” comprises anysubsequent generation of a plant, plant cell, plant tissue, or plantorgan.

Furthermore, the term “derivative” can imply, in the context of asubstance or molecule rather than referring to a cell or organism,directly or by means of modification indirectly obtained from another.This might imply a nucleic acid sequence derived from a cell or a plantmetabolite obtained from a cell or material.

Furthermore, the terms “derived”, or “derived from” as used herein inthe context of a biological sequence (nucleic acid or amino acid) or amolecule or a complex imply that the respective sequence is based on areference sequence, for example from the sequence listing, or a databaseaccession number, or the respective scaffold structure, i.e.,originating from said sequence, whereas the reference sequence cancomprise more sequences, e.g., the whole genome or a full polyproteinencoding sequence, of a virus, whereas the sequence “derived from” thenative sequence may only comprise one isolated fragment thereof, or acoherent fragment thereof. In this context, a cDNA molecule or a RNA canbe said to be “derived from” a DNA sequence serving as moleculartemplate. The skilled person can thus easily define a sequence “derivedfrom” a reference sequence, which will, by sequence alignment on DNA oramino acid level, have a high identity to the respective referencesequence and which will have coherent stretches of DNA/amino acids incommon with the respective reference sequence (>75% query identity for agiven length of the molecule aligned provided that the derived sequenceis the query and the reference sequence represents the subject during asequence alignment). The skilled person can thus clone the respectivesequences based on the disclosure provided herein by means of polymerasechain reactions and the like into a suitable vector system of interest,or use a sequence as vector scaffold. The term “derived from” is thus noarbitrary sequence, but a sequence corresponding to a reference sequenceit is derived from, whereas certain differences, e.g., certain mutationsnaturally occurring during replication of a recombinant construct withina host cell, cannot be excluded and are thus comprised by the term“derived from”. Furthermore, several sequence stretches from a parentsequence can be concentrated in a sequence derived from the parent. Thedifferent stretches will have high or even 100% homology to the parentsequence.

The term an “endogenous” in the context of nucleic acid and/or aminoacid sequences refers to the nucleic acid and/or amino acid as found ina plant genome in its natural form and natural genetic context. As it isknown to the skilled person, several variants, e.g., allelic variants,of a gene nucleic acid sequence may exist in a given species of plants.

A “fungus” or “fungal pathogen” as used herein means any plantpathogenic fungus including oomycetes in any developmental stage,including spores, or any part of such a fungus, which can interact witha plant or plant part or cell to induce a response in said plant orplant part or cell.

As used herein, “fusion” can refer to a protein and/or nucleic acidcomprising one or more non-native sequences (e.g., moieties). A fusioncan be at the N-terminal or C-terminal end of the modified protein, orboth, or within the molecule as separate domain. For nucleic acidmolecules, the fusion molecule can be attached at the 5′ or 3′ end, orat any suitable position in between. A fusion can be a transcriptionaland/or translational fusion. A fusion can comprise one or more of thesame non-native sequences. A fusion can comprise one 10 or more ofdifferent non-native sequences. A fusion can be a chimera. A fusion cancomprise a nucleic acid affinity tag. A fusion can comprise a barcode. Afusion can comprise a peptide affinity tag. A fusion can provide forsubcellular localization of the site-specific effector or base editor(e.g., a nuclear localization signal (NLS) for targeting to the nucleus,a mitochondrial localization signal for targeting to the mitochondria, achloroplast localization signal for targeting to a chloroplast, anendoplasmic reticulum (ER) retention signal, and the like). A fusion canprovide a non-native sequence (e.g., affinity tag) that can be used totrack or purify. A fusion can be a small molecule such as biotin or adye such as alexa fluor dyes, Cyanine3 dye, Cyanine5 dye. The fusion canprovide for increased or decreased stability. In some embodiments, afusion can comprise a detectable label, including a moiety that canprovide a detectable signal. Suitable detectable labels and/or moietiesthat can provide a detectable signal can include, but are not limitedto, an enzyme, a radioisotope, a member of a specific binding pair; afluorophore; a fluorescent reporter or fluorescent protein; a quantumdot; and the like. A fusion can comprise a member of a FRET pair, or afluorophore/quantum dot donor/acceptor pair. A fusion can comprise anenzyme. Suitable enzymes can include, but are not limited to, horseradish peroxidase, luciferase, beta-25 galactosidase, and the like. Afusion can comprise a fluorescent protein. Suitable fluorescent proteinscan include, but are not limited to, a green fluorescent protein (GFP),(e.g., a GFP from Aequoria victoria, fluorescent proteins from Anguillajaponica, or a mutant or derivative thereof), a red fluorescent protein,a yellow fluorescent protein, a yellow-green fluorescent protein (e.g.,mNeonGreen derived from a tetrameric fluorescent protein from thecephalochordate Branchiostoma lanceolatum) any of a variety offluorescent and colored proteins. A fusion can comprise a nanoparticle.Suitable nanoparticles can include fluorescent or luminescentnanoparticles, and magnetic nanoparticles, or nanodiamonds, optionallylinked to a nanoparticle. Any optical or magnetic property orcharacteristic of the nanoparticle(s) can be detected. A fusion cancomprise a helicase, a nuclease (e.g., Fok1), an endonuclease, anexonuclease (e.g., a 5′ exonuclease and/or 3′ exonuclease), a ligase, anickase, a nuclease-helicase (e.g., Cas3), a DNA methyltransferase(e.g., Dam), or DNA demethylase, a histone methyltransferase, a histonedemethylase, an acetylase (including for example and not limitation, ahistone acetylase), a deacetylase (including for example and notlimitation, a histone deacetylase), a phosphatase, a kinase, atranscription (co-) activator, a transcription (co-) factor, an RNApolymerase subunit, a transcription repressor, a DNA binding protein, aDNA structuring protein, a long non-coding RNA, a DNA repair protein(e.g., a protein involved in repair of either single- and/ordouble-stranded breaks, e.g., proteins involved in base excision repair,nucleotide excision repair, mismatch repair, NHEJ, HR,microhomology-mediated end joining (MMEJ), and/or alternativenon-homologous end-joining (ANHEJ), such as for example and notlimitation, HR regulators and HR complex assembly signals), a markerprotein, a reporter protein, a fluorescent protein, a ligand bindingprotein (e.g., mCherry or a heavy metal binding protein), a signalpeptide (e.g., Tat-signal sequence), a targeting protein or peptide, asubcellular localization sequence (e.g., nuclear localization sequence,a chloroplast localization sequence), and/or an antibody epitope, or anycombination thereof.

The term “genetically modified” or “genetic manipulation” or“genetic(ally) manipulated” is used in a broad sense herein and meansany modification of a nucleic acid sequence or an amino acid sequence, atarget cell, tissue, organ or organism, which is accomplished by humanintervention, either directly or indirectly, to influence the endogenousgenetic material or the transciptome or the proteome of a target cell,tissue, organ or organism to modify it in a purposive way so that itdiffers from its state as found without human intervention. The humanintervention can either take place in vitro or in vivo/in planta, oralso both. Further modifications can be included, for example, one ormore point mutation(s), e.g. for targeted protein engineering or forcodon optimization, deletion(s), and one or more insertion(s) ordeletion(s) of at least one nucleic acid or amino acid molecule(including also homologous recombination), modification of a nucleicacid or an amino acid sequence, or a combination thereof. The termsshall also comprise a nucleic acid molecule or an amino acid molecule ora host cell or an organism, including a plant or a plant materialthereof which is/are similar to a comparable sequence, organism ormaterial as occurring in nature, but which have been constructed by atleast one step of purposive manipulation. A “targeted geneticmanipulation” or “targeted (base) modification” as used herein is thusthe result of a “genetic manipulation”, which is effected in a targetedway, i.e. at a specific position in a target cell and under the specificsuitable circumstances to achieve a desired effect in at least one cell,preferably a plant cell, to be manipulated, wherein the term impliesthat the sequence to be targeted and the corresponding modification arebased on preceding sequence considerations so that the resultingmodification can be planned in advance, e.g., based on availablesequence information of a target site in the genome of a cell and/orbased on the information of the target specificity (recognition orbinding properties of a nucleic acid or an amino acid sequence,complementary base pairing and the like) of a molecular tool ofinterest.

The term “genome” refers to the entire complement of genetic material(genes and non-coding sequences) that is present in each cell of anorganism, or virus or organelle, and/or a complete set of chromosomesinherited as a (haploid) unit from one parent. The genome thus alsodefines the “genotype” being the part of the genetic makeup of a givencell, and therefore of an organism or individual, which determines aspecific characteristic (phenotype) of that cell/organism/individual.

The terms “genome editing”, “genome engineering”, or “geneediting/engineering” are used interchangeably herein and refer tostrategies and techniques for the targeted, specific modification of anygenetic information or genome of a living organism. As such, the termscomprise gene editing, but also the editing of regions other than geneencoding regions of a genome. It further comprises the editing orengineering of the nuclear (if present) as well as other geneticinformation of a cell. Furthermore, the terms “genome editing” and“genome engineering” also comprise an epigenetic editing or engineering,i.e., the targeted modification of, e.g., methylation, histonemodification or of non-coding RNAs possibly causing heritable changes ingene expression.

“Germplasm”, as used herein, is a term used to describe the geneticresources, or more precisely the DNA of an organism and collections ofthat material. In breeding technology, the term germplasm is used toindicate the collection of genetic material from which a new plant orplant variety can be created.

The terms “guide RNA”, “gRNA” or “single guide RNA” or “sgRNA” are usedinterchangeably herein and either refer to a synthetic fusion of aCRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), or the termrefers to a single RNA molecule consisting only of a crRNA and/or atracrRNA, or the term refers to a gRNA individually comprising a crRNAor a tracrRNA moiety. The tracr and the crRNA moiety thus do notnecessarily have to be present on one covalently attached RNA molecule,yet they can also be comprised by two individual RNA molecules, whichcan associate or can be associated by non-covalent or covalentinteraction to provide a gRNA according to the present disclosure. Theterms “gDNA” or “sgDNA” or “guide DNA” are used interchangeably hereinand either refer to a nucleic acid molecule interacting with anArgonaute nuclease. Both, the gRNAs and gDNAs as disclosed herein aretermed “guiding nucleic acids” or “guide nucleic acids” due to theircapacity to interacting with a site-specific nuclease and to assist intargeting said site-specific nuclease to a genomic target site.

The term “hybridization” as used herein refers to the pairing ofcomplementary nucleic acids, i.e., DNA and/or RNA, using any process bywhich a strand of nucleic acid joins with a complementary strand throughbase pairing to form a hybridized complex. Hybridization and thestrength of hybridization (i.e., the strength of the association betweenthe nucleic acids) is impacted by such factors as the degree and lengthof complementarity between the nucleic acids, stringency of theconditions involved, the Tm of the formed hybrid, and the G:C ratiowithin the nucleic acids. The term hybridized complex refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bounds between complementary G and C bases andbetween complementary A and T/U bases. A hybridized complex or acorresponding hybrid construct can be formed between two DNA nucleicacid molecules, between two RNA nucleic acid molecules or between a DNAand an RNA nucleic acid molecule. For all constellations, the nucleicacid molecules can be naturally occurring nucleic acid moleculesgenerated in vitro or in vivo and/or artificial or synthetic nucleicacid molecules. Hybridization as detailed above, e.g., Watson-Crick basepairs, which can form between DNA, RNA and DNA/RNA sequences, aredictated by a specific hydrogen bonding pattern, which thus represents anon-covalent attachment form according to the present invention. In thecontext of hybridization, the term “stringent hybridization conditions”should be understood to mean those conditions under which ahybridization takes place primarily only between homologous nucleic acidmolecules. The term “hybridization conditions” in this respect refersnot only to the actual conditions prevailing during actual agglomerationof the nucleic acids, but also to the conditions prevailing during thesubsequent washing steps. Examples of stringent hybridization conditionsare conditions under which primarily only those nucleic acid moleculesthat have at least at least 80%, preferably at least 85%, at least 90%or at least 95% sequence identity undergo hybridization. Stringenthybridization conditions are, for example: 4×SSC at 65° C. andsubsequent multiple washes in 0.1×SSC at 65° C. for approximately 1hour. The term “stringent hybridization conditions” as used herein mayalso mean: hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2,7% SDS, 1 mM EDTA and 1% BSA for 16 hours and subsequently washing twicewith 2×SSC and 0.1% SDS at 68° C. Preferably, hybridization takes placeunder stringent conditions.

The term “introgression” as used herein refers to the transfer of atleast one allele of a gene of interest on a genetic locus from onegenetic background to another. For example, introgression can proceedthrough sexual crossing of two parents of the same species.Alternatively, the transfer of a gene allele can take place byrecombination between two donor genomes, e.g., in a fused protoplast,wherein at least the donor protoplast carries the gene allele ofinterest in its genome. In any case, any progeny or derivativescomprising the gene allele of interest can then be subjected to repeatedback-crossing steps with a plant line carrying a genetic background ofinterest to select for the gene allele of interest in the resultingderivatives or progeny. The result may be the fixation of the geneallele of interest such introgressed in a selected genetic background.The whole process of introgression can, for example, take place by amixture of breeding strategies and techniques of molecular biology toachieve at a genotype/phenotype of interest for a given germplasm,plant, plant cell or plant material.

The term “locus” generally refers to a genetically defined region of achromosome carrying a gene or, possibly, two or more genes so closelylinked that genetically they behave as a single locus responsible for aphenotype.

As used herein, the terms “mutation” and “modification” are usedinterchangeably to refer to a deletion, insertion, addition,substitution, edit, strand break, and/or introduction of an adduct inthe context of nucleic acid manipulation in vivo or in vitro. A deletionis defined as a change in a nucleic acid sequence in which one or morenucleotides is absent. An insertion or addition is that change in anucleic acid sequence which has resulted in the addition of one or morenucleotides. A “substitution” or “edit” results from the replacement ofone or more nucleotides by a molecule which is a different molecule fromthe replaced one or more nucleotides. For example, a nucleic acid may bereplaced by a different nucleic acid as exemplified by replacement of athymine by a cytosine, adenine, guanine, or uridine. Pyrimidine topyrimidine (e.g., C to Tor T to C nucleotide substitutions) or purine topurine (e.g., G to A or A to G nucleotide substitutions) are termedtransitions, whereas pyrimidine to purine or purine to pyrimidine (e.g.,G to T or G to C or A to T or A to C) are termed transversions.Alternatively, a nucleic acid may be replaced by a modified nucleic acidas exemplified by replacement of a thymine by thymine glycol. Mutationsmay result in a mismatch. The term mismatch refers to a non-covalentinteraction between two nucleic acids, each nucleic acid residing on adifferent nucleotide sequence or nucleic acid molecule, which does notfollow the base-pairing rules. For example, for the partiallycomplementary sequences 5′-AGT-3′ and 5′-AAT-3′, a G-A mismatch (atransition) is present.

“Near isogenic lines” or “NILs” as used herein are useful foridentifying genes responsible for a phenotypic trait by mapping them togenetic chromosomes by analyzing NILs. To create a near isogenic line,an organism with the phenotype of interest, often a plant, is crossedwith a standard line of the same plant. The F1 generation is selfed toproduce the F2 generation. F2 individuals with the target trait areselected for crossing with the standard line (the recurrent parent).This process is repeated for several generations. The genetic make-upsof sister lines can be compared. Alleles derived from the donor parentthat can be found in all sister lines are said to be associated with thetrait.

The terms “nucleotide” and “nucleic acid” with reference to a sequenceor a molecule are used interchangeably herein and refer to a single- ordouble-stranded DNA or RNA of natural or synthetic origin. The termnucleotide sequence is thus used for any DNA or RNA sequence independentof its length, so that the term comprises any nucleotide sequencecomprising at least one nucleotide, but also any kind of largeroligonucleotide or polynucleotide. The term(s) thus refer to naturaland/or synthetic deoxyribonucleic acids (DNA) and/or ribonucleic acid(RNA) sequences, which can optionally comprise synthetic nucleic acidanaloga. A nucleic acid according to the present disclosure canoptionally be codon optimized “Codon optimization” implies that thecodon usage of a DNA or RNA is adapted to that of a cell or organism ofinterest to improve the transcription rate of said recombinant nucleicacid in the cell or organism of interest. The skilled person is wellaware of the fact that a target nucleic acid can be modified at oneposition due to the codon degeneracy, whereas this modification willstill lead to the same amino acid sequence at that position aftertranslation, which is achieved by codon optimization to take intoconsideration the species-specific codon usage of a target cell ororganism. Nucleic acid sequences according to the present applicationcan carry specific codon optimization for the following non limitinglist of organisms: Hordeum vulgare, Sorghum bicolor, Secale cereale,Saccharum officinarium, Zea mays, Setaria italic, Oryza sativa, Oryzaminuta, Oryza australiensis, Oryza alta, Triticum aestivum, Triticumdurum, Triticale, Hordeum bulbosum, Brachypodium distachyon, Hordeummarinum, Aegilops tauschii, Ma/us domestica, Beta vulgaris, Helianthusannuus, Daucus glochidiatus, Daucus pusillus, Daucus muricatus, Daucuscarota, Eucalyptus grandis, Erythranthe guttata, Genlisea aurea,Nicotiana sylvestris, Nicotiana tabacum, Nicotiana tomentosiformis,Nicotiana benthamiana, Solanum/ycopersicum, Solanum tuberosum, Coffeacanephora, Vitis vinifera, Cucumis sativus, Marus notabilis, Arabidopsisthaliana, Arabidopsis lyrata, Arabidopsis arenosa, Crucihimalayahimalaica, Crucihimalaya wallichii, Cardamine flexuosa, Lepidiumvirginicum, Capsella bursa-pastoris, Olmarabidopsis pumila, Arabishirsuta, Brassica napus, Brassica oleracea, Brassica rapa, Brassicajuncacea, Brassica nigra, Raphanus sativus, Eruca vesicaria sativa,Citrus sinensis, Jatropha curcas, Glycine max, Gossypium ssp., orPopulus trichocarpa

The term “particle bombardment” as used herein, also named “biolistictransfection” or “microparticle-mediated gene transfer”, refers to aphysical delivery method for transferring a coated microparticle ornanoparticle comprising a nucleic acid or a genetic construct ofinterest into a target cell or tissue. The micro or nanoparticlefunctions as projectile and is fired on the target structure of interestunder high pressure using a suitable device, often called gene-gun. Thetransformation via particle bombardment uses a microprojectile of metalcovered with the gene of interest, which is then shot onto the targetcells using an equipment known as “gene gun” (Sanford, John C., et al.“Delivery of substances into cells and tissues using a particlebombardment process.” Particulate Science and Technology 5.1 (1987):27-37) at high velocity fast enough (˜1500 km/h) to penetrate the cellwall of a target tissue, but not harsh enough to cause cell death. Forprotoplasts, which have their cell wall entirely removed, the conditionsare different logically. The precipitated nucleic acid or the geneticconstruct on the at least one microprojectile is released into the cellafter bombardment, and integrated into the genome. The acceleration ofmicroprojectiles is accomplished by a high voltage electrical dischargeor compressed gas (helium). Concerning the metal particles used it ismandatory that they are non-toxic, non-reactive, and that they have alower diameter than the target cell. The most commonly used are gold ortungsten. There is plenty of information publicly available from themanufacturers and providers of gene-guns and associated systemconcerning their general use.

A “pathogen” as used herein refers to an organism which can infect aplant, or which can cause a disease in a plant. Pathogens which caninfect a plant, or which can cause a disease in a plant, include fungi,oomycetes, bacteria, viruses, viroids, virus-like organisms,phytoplasmas, protozoa, nematodes and parasitic plants. Plant parasitescan cause damage by feeding on a plant and can be selected fromectoparasites like insects, comprising aphids and other sap-suckinginsect, mites, and vertebrates.

The term “plant” as used herein is to be construed broadly and refers toa whole plant organism, a plant organ, differentiated andundifferentiated plant tissues, plant cells, seeds, and derivatives andprogeny thereof. “Plant cells” include without limitation, for example,cells from seeds, from mature and immature embryos, meristematictissues, seedlings, callus tissues in different differentiation states,leaves, flowers, roots, shoots, gametophytes, grains, kernels,sporophytes, pollen and microspores, protoplasts, macroalgae andmicroalgae. The different plant cells can either be haploid, diploid ormultiploid. The term “plant organ” refers to plant tissue or a group oftissues that constitute a morphologically and functionally distinct partof a plant. Typically, the term “grain” is used to describe the maturekernel produced by a plant grower for purposes other than growing orreproducing the species, and “seed” means the mature kernel used forgrowing or reproducing the species. For the purposes of the presentinvention, “grain”, “seed”, and “kernel”, will be used interchangeably.

A “plant material” as used herein refers to any material which can beobtained from a plant during any developmental stage. The plant materialcan be obtained either in planta or from an in vitro culture of theplant or a plant tissue or organ thereof. The term thus comprises plantcells, tissues and organs as well as developed plant structures as wellas sub-cellular components like nucleic acids, polypeptides and allchemical plant substances or metabolites which can be found within aplant cell or compartment and/or which can be produced by the plant, orwhich can be obtained from an extract of any plant cell, tissue or aplant in any developmental stage. The term also comprises a derivativeof the plant material, e.g., a protoplast, derived from at least oneplant cell comprised by the plant material. The term therefore alsocomprises meristematic cells or a meristematic tissue of a plant.

A “control” or “control plant cell” or “control tissue” or “controlorgan” or “control plant” provides reference point for measuring changesin phenotype of a subject plant or plant part in which (genetic)modification, modulation and/or alteration, such as indicated in thevarious aspects of the present invention, has been affected to a gene, aprotein or a substance or molecule of interest. A control plant orcontrol plant part (e.g. control plant cell, control tissue, or controlorgan) may comprise, for example: (a) a wild-type plant or cell, i.e.,of the same genotype as the starting material for the (genetic)modification, modulation and/or alteration which resulted in the subjectplant or plant part; (b) a plant or plant part of the same genotype asthe starting material but which has been transformed with a nullconstruct (i.e., with a construct which has no known effect on the traitof interest, such as a construct comprising a marker gene); or (c) aplant or plant part which is a non-transformed segregant among progenyof a subject plant or plant part. In particular, a control plant orcontrol plant cell may comprise a plant or plant part of the samegenotype, but lacking the modification of the at least one gene encodingat least one wall-associated kinase or the modulation of the expressionlevel of at least one wall-associated kinase and/or the transcriptionlevel, the expression level, or the function of at least one moleculewithin the signaling pathway from the at least one wall-associatedkinase to the synthesis of at least one benzoxazinoid or within thesynthesis pathway of at least one benzoxazinoid.

A “plasmid” refers to a circular autonomously replicatingextrachromosomal element in the form of a double-stranded nucleic acidsequence. In the field of genetic engineering these plasmids areroutinely subjected to targeted modifications by inserting, for example,genes encoding a resistance against an antibiotic or an herbicide, agene encoding a target nucleic acid sequence, a localization sequence, aregulatory sequence, a tag sequence, a marker gene, including anantibiotic marker or a fluorescent marker, and the like. The structuralcomponents of the original plasmid, like the origin of replication, aremaintained. According to certain embodiments of the present invention,the localization sequence can comprise a nuclear localization sequence,a plastid localization sequence, preferably a mitochondrion localizationsequence or a chloroplast localization sequence, or a localizationsequence for targeting a kinase of interest to the plasma membrane of acell of interest. Said localization sequences are available to theskilled person in the field of plant biotechnology. A variety of plasmidvectors for use in different target cells of interest is commerciallyavailable and the modification thereof is known to the skilled person inthe respective field.

The terms “protein”, “amino acid” or “polypeptide” are usedinterchangeably herein and refer to an amino acid sequence having acatalytic enzymatic function or a structural or a functional effect. Theterm “amino acid” or “amino acid sequence” or “amino acid molecule”comprises any natural or chemically synthesized protein, peptide,polypeptide and enzyme or a modified protein, peptide, polypeptide andenzyme, wherein the term “modified” comprises any chemical or enzymaticmodification of the protein, peptide, polypeptide and enzyme, includingtruncations of a wild-type sequence to a shorter, yet still activeportion. In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S J. Higgins eds.(1985); Transcription and Translation (B. D. Hames & S. J. Higgins, eds.(1984); among others.

The term “regulatory sequence” or “regulatory region” as used hereinrefers to a nucleic acid or an amino acid sequence, which can directand/or influence the transcription and/or translation and/ormodification of a nucleic acid sequence of interest. A regulatorysequence may be a promoter sequence, an enhancer, a silencer, atranscription factor and the like.

The terms “resistance” or “tolerance” or “resistant” or “tolerant” asused herein refers to the capacity of a plant to resist to the phenotypeas caused by infestation with a pathogen, in particular a fungalpathogen, disclosed herein to a certain degree, i.e., the prevention,reduction or delay of an infection caused by a (fungal) pathogen.“Resistance”/“Tolerance”, therefore, does not exclusively refer to a“black or white” phenotype implying a phenotype where no symptoms occurat all after infestation for a resistant plant. The terms “resistance”or “tolerance” or “resistant” or “tolerant” rather imply a gradualimprovement of infestation symptoms as observed for a plant having noresistance to a given pathogen. A classification score scheme forphenotyping experiments in field trials at various locations withnatural and artificial H. turcicum inoculation (from the DeutscheMaiskomitee (DMK, German maize committee); AG variety 27.02.02; (DMK J.Rath; R P Freiburg H. J. Imgraben) shows a resistance level from 9 (low)to 1 (high) for maize as exemplary plant, wherein each score representsthe following phenotype: 1: Plants exhibit no symptoms of disease, 0%;2: Beginning of infestation, first small spots (less than 2 cm) visible.Less than 5% of leaf surface affected. 3: Some spots have developed on aleaf stage. Between 5-10% of leaf surface affected. 4: 10-20% of leafsurface affected. Clearly visible spots on several leaf stages. 5:20-40% of leaf surface affected. Spots start to coalesce. 6: 40-60% ofleaf surface affected. Systematic infestation visible on leaves. 7:60-80% of leaf surface affected. Approximately half of leaves destroyedor dried out because of fungal infestation. 8: 80-90% of leaf surfaceaffected. More than half of leaves destroyed or dried out because offungal infestation. 9: 90-100% of leaf surface affected. The plants arealmost completely dried out.

The term “TILLING” as used herein is an abbreviation for “TargetingInduced Local Lesions in Genomes” and describes a well-known reversegenetics technique designed to detect unknown SNPs (single nucleotidepolymorphisms) in genes of interest using an enzymatic digestion and iswidely employed in plant genomics. The technique allows for thehigh-throughput identification of an allelic series of mutants with arange of modified functions for a particular gene. TILLING combinesmutagenesis (e.g., chemical or via UV-light) with a sensitive DNAscreening-technique that identifies single base mutations.

The terms “transgene” or “transgenic” as used herein refer to at leastone nucleic acid sequence that is taken from the genome of one organism,or produced synthetically, and which is then introduced into a host cellor organism or tissue of interest and which is subsequently integratedinto the host's genome by means of “stable” transformation ortransfection approaches. In contrast, the term “transient”transformation or transfection or introduction refers to a way ofintroducing molecular tools including at least one nucleic acid(comprising at least one of DNA, RNA, single-stranded or double-strandedor a mixture thereof) and/or at least one amino acid sequence,optionally comprising suitable chemical or biological agents, to achievea transfer into at least one compartment of interest of a cell,including, but not restricted to, the cytoplasm, an organelle, includingthe nucleus, a mitochondrion, a vacuole, a chloroplast, or into amembrane, resulting in transcription and/or translation and/orassociation and/or activity of the at least one molecule introducedwithout achieving a stable integration or incorporation and thusinheritance of the respective at least one molecule introduced into thegenome of a cell.

The term “transient introduction” as used herein thus refers to thetransient introduction of at least one nucleic acid and/or amino acidsequence according to the present disclosure, preferably incorporatedinto a delivery vector or into a recombinant construct, with or withoutthe help of a delivery vector, into a target structure, for example, aplant cell, wherein the at least one nucleic acid sequence is introducedunder suitable reaction conditions so that no integration of the atleast one nucleic acid sequence into the endogenous nucleic acidmaterial of a target structure, the genome as a whole, occurs, so thatthe at least one nucleic acid sequence will not be integrated into theendogenous DNA of the target cell. As a consequence, in the case oftransient introduction, the introduced genetic construct will not beinherited to a progeny of the target structure, for example aprokaryotic or a plant cell. The at least one nucleic acid and/or aminoacid sequence or the products resulting from transcription, translation,processing, post-translational modifications or complex building thereofare only present temporarily, i.e., in a transient way, in constitutiveor inducible form, and thus can only be active in the target cell forexerting their effect for a limited time. Therefore, the at least onesequence or effector introduced via transient introduction will not beheritable to the progeny of a cell. The effect mediated by at least onesequence or effector introduced in a transient way can, however,potentially be inherited to the progeny of the target cell.

A “variant” in the context of a nucleic acid or amino acid sequenceprotein means a nucleic acid or amino acid sequence derived from thenative nucleic acid or amino acid sequence, or another startingsequence, by deletion (so-called truncation) or addition of one or moresequences to the 5′/N-terminal and/or 3′/C-terminal end of the nativenucleic acid or amino acid sequence; deletion or addition of one or morenucleic acid or amino acid sequence at one or more sites in the nativenucleic acid or amino acid sequence; or substitution of one or morenucleic acid or amino acid sequence at one or more sites in the nativeprotein. Variant proteins encompassed by the present invention arebiologically active, that is they continue to possess all or some of theactivity of the native proteins of the invention as described herein.Such variants may result from, for example, genetic polymorphism or fromhuman manipulation.

As used herein, a “homolog” means a protein in a group of proteins thatperform the same biological function, e.g. proteins that belong to thesame Pfam protein family. Homologs are expressed by homologous genes.With reference to homologous genes, homologs include orthologs, e.g.,genes expressed in different species that evolved from a commonancestral genes by speciation and encode proteins retain the samefunction, but do not include paralogs, e.g., genes that are related byduplication but have evolved to encode proteins with differentfunctions. Homologous genes include naturally occurring alleles andartificially-created variants. Degeneracy of the genetic code providesthe possibility to substitute at least one base of the protein encodingsequence of a gene with a different base without causing the amino acidsequence of the polypeptide produced from the gene to be changed. Whenoptimally aligned, homolog proteins have typically at least about 60%identity, in some instances at least about 70%, for example about 80% or85% and even at least about 90%, 92%, 94%, 96%, 97%, 98%, 99% or 99.5%identity, preferably over the full length of the protein; homologousgenes have typically at least about 60% identity, in some instances atleast about 70%, for example about 80% or 85% and even at least about90%, 92%, 94%, 96%, 97%, 98%, 99% or 99.5% identity, preferably over thefull length of the gene, in particular the coding regions of the gene.Homologs are identified by comparison of amino acid sequence, e.g.manually or by use of a computer-based tool using known homology-basedsearch algorithms such as those commonly known and referred to as BLAST,FASTA, and Smith-Waterman. A local sequence alignment program, e.g.BLAST, can be used to search a database of sequences to find similarsequences, and the summary Expectation value (E-value) used to measurethe sequence base similarity. Because a protein hit with the bestE-value for a particular organism may not necessarily be an ortholog,e.g., have the same function, or be the only ortholog, a reciprocalquery is used to filter hit sequences with significant E-values forortholog identification. The reciprocal query entails search of thesignificant hits against a database of amino acid sequences from thebase organism that are similar to the sequence of the query protein. Ahit can be identified as an ortholog, when the reciprocal query's besthit is the query protein itself or a protein encoded by a duplicatedgene after speciation. A further aspect of the homologs encoded by DNAuseful in the transgenic plants of the invention are those proteins thatdiffer from a disclosed protein as the result of deletion or insertionof one or more amino acids in a native sequence.

Other functional homolog proteins differ in one or more amino acids fromthose disclosed herein as the result of one or more of the well-knownconservative amino acid substitutions, e.g., valine is a conservativesubstitute for alanine and threonine is a conservative substitute forserine. Conservative substitutions for an amino acid within the nativesequence can be selected from other members of a class to which thenaturally occurring amino acid belongs. Representative amino acidswithin these various classes include, but are not limited to: (1) acidic(negatively charged) amino acids such as aspartic acid and glutamicacid; (2) basic (positively charged) amino acids such as arginine,histidine, and lysine; (3) neutral polar amino acids such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and(4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.Conserved substitutes for an amino acid within a native amino acidsequence can be selected from other members of the group to which thenaturally occurring amino acid belongs. For example, a group of aminoacids having aliphatic side chains is glycine, alanine, valine, leucine,and isoleucine; a group of amino acids having aliphatic-hydroxyl sidechains is serine and threonine; a group of amino acids havingamide-containing side chains is asparagine and glutamine; a group ofamino acids having aromatic side chains is phenylalanine, tyrosine, andtryptophan; a group of amino acids having basic side chains is lysine,arginine, and histidine; and a group of amino acids havingsulfur-containing side chains is cysteine and methionine. Naturallyconservative amino acids substitution groups are: valine-leucine,valine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. Afurther aspect of the invention includes proteins that differ in one ormore amino acids from those of a described protein sequence as theresult of deletion or insertion of one or more amino acids in a nativesequence.

Whenever the present disclosure relates to the percentage of thehomology or identity of nucleic acid or amino acid sequences thesevalues define those as obtained by using the EMBOSS Water PairwiseSequence Alignments (nucleotide) programme(www.ebi.ac.uk/Tools/psa/emboss_water/nucleotide.html) nucleic acids orthe EMBOSS Water Pairwise Sequence Alignments (protein) programme(www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences,preferably over the entire length of the sequence, i.e., any percentagevalue provided means the % homology or % identity as measured over thewhole length of a subject or starting sequence in comparison to anidentical or variant further sequence. Those tools provided by theEuropean Molecular Biology Laboratory (EMBL) European BioinformaticsInstitute (EBI) for local sequence alignments use a modifiedSmith-Waterman algorithm (see www.ebi.ac.uk/Tools/psa/ and Smith, T. F.& Waterman, M. S. “Identification of common molecular subsequences”Journal of Molecular Biology, 1981 147 (1):195-197). When conducting analignment, the default parameters defined by the EMBL-EBI are used.Those parameters are (i) for amino acid sequences: Matrix=BLOSUM62, gapopen penalty=10 and gap extend penalty=0.5 or (ii) for nucleic acidsequences: Matrix=DNAfull, gap open penalty=10 and gap extendpenalty=0.5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (FIGS. 1 A and B) shows (FIG. 1 A) the structure of benzoxazinone(BXD) in the aglucone form. Derivatives from the BXD basic structure ofFIG. 1 A are: HBOA (R¹═H, R²═H, R³═H), DHBOA (R¹═H, R²═OH, R³═H), HMBOA(R¹═H, R²═OMe, R³═H), HM₂BOA (R¹═H, R²═OMe, R³═OMe), DIBOA (R¹═OH, R²═H,R³═H), TRIBOA (R¹═OH, R²═OH, R³═H), DIMBOA (R¹═OH, R²═OMe, R³═H),DIM₂BOA (R¹═OH, R²═OMe, R³═OMe), HDMBOA (R¹═OMe, R²═OMe, R³═H), orHDM2BOA (R¹═OMe, R²═OMe, R³═OMe). FIG. 1 B shows the basic structure ofa benzoxazolinone, a natural degradation product of BXDs and alsocomprised by the term BXD as used herein. Specific benzoxazolinones areBOA (R²═H, R³═H), MBOA (R²═OMe, R³═H), or M₂BOA (R²═OMe, R³═OMe).

FIG. 2 (FIGS. 2 A and B) shows the rate of successful penetration eventsfor fungal penetrations as described in Example 9. (FIG. 2 A) Hyphaedetected inside of host tissues. In the upper and lower panels the focusis on the epidermis and hyphae, respectively. The arrows in the lowerimage of Fig. A indicate the hyphae inside the host tissues. (FIG. 2 B)RLK1b, RLK1d and RLK1f are ZmWAK-RLK1 mutants with compromised NCLBresistance that were produced in RP3Htn1, while RLK1b-wt, RLK1d-wt andRLK1f-wt are the corresponding sister lines, respectively. Statisticswas conducted using Student's t test, based on three independentexperiments. The asterisks represent a significant difference of**p<0.01 or *p<0.05. Error bars indicate ±standard error.

FIG. 3 (FIG. 3 A to C) shows the disease phenotype of Htn1-NILs and thecorresponding parents as detailed in Example 10 below. (FIG. 3 A) Thedisease symptom of the second leaves at 16 dpi (from left to right forthe lines w22, w22Htn1, B37 and B37Htn1). (FIG. 3 B) Rate of infected oftested plants B37, w22, w22Htn1, and B37Htn1. (FIG. 3 C) Area under thedisease progress curve (AUDPC) for the isogenic lines detailed below thex-axis. *AUDPC in this panel was calculated as described in Hurni et al.2015, based on calculating sum of rate of infected plants (%).

FIG. 4 shows the result of a transcriptome analysis in Htn1-NILs, whichrevealed a set of DEGs. FIG. 4 shows the number of DEGs in the two Htn1NILs compared to the corresponding susceptible lines as furtherdescribed in Example 10 below.

FIG. 5 (FIG. 5 A to F) shows the content of BXDs in w22 and w22Htn1.(FIG. 5 A) shows the proposed bio-synthesis pathway of secondarymetabolites BXDs. The genes encoding the proteins catalyzing each stepof enzymatic reactions are presented besides and below the arrows. Thecontents of BXDs compounds DIMBOA-Glc (FIG. 5 B), DIMBOA (FIG. 5 C),HMBOA-Glc (FIG. 5 D), DIM₂BOA-Glc (FIG. 5 E) and HDMBOA-Glc (FIG. 5 F)were determined at before inoculation, at 3 dpi and 10 dpi. Thestatistics were conducted using Tukey's HSD (P=0.05) in eight biologicalreplicates. The ns stands for no significance. Error bars are ±SE. Seealso Example 10 below.

FIG. 6 (FIG. 6 A to D) shows that the presence of ZmWAK-RLK1 results indecreased DIM₂BOA-Glc content possibly by down-regulating Igl expressionin the maize RP3 genetic background (see also Example 11). (A) Contentof DIM₂BOA-Glc (n=8). Expression analysis of Bx1 (B), Igl (C) andZmWAK-RLK1 (D) in Htn1 mutants and sister lines before infection at 21days after sowing (n=5). Statistics was conducted using Student's ttest. The asterisks represent a significant difference of **p<0.01 or*p<0.05. Error bars are ±SE.

FIG. 7 (FIG. 7 A to E) shows the content of BXDs compounds DIMBOA-Glc(A), DIMBOA (B), HMBOA-Glc (C), and DIM₂BOA-Glc (D), HDMBOA-Glc (E) insecond leaves at 10 dpi. Further information is provided in Example 12below. The statistics were conducted using Student's t test (P=0.05) ineight biological replicates. The asterisks represent a significantdifference of **p<0.01 or *p<0.05. The ns stands for no significance.Error bars are ±SE.

FIG. 8 (FIG. 8 A to D) (see also Example 12) shows that compromising thebiosynthesis of BXDs decreases the susceptibility of NCLB disease at theseedling stage. (FIGS. 8 A and B): Visual symptoms and quantified NCLBdisease severity in bx mutants. (FIG. 8 C): The transcriptional level ofZmWAK-RLK1 at 10 dpi. (FIG. 8 D) shows the proposed model of ZmWAK-RLK1underlying NCLB disease resistance. The resistance allele suppressed thebiosynthesis of major BXDs compounds, which likely served as thesusceptibility component for promoting NCLB disease. Statistic test wasconducted using Student's t test. The asterisks represent a significantdifference of **p<0.01 or *p<0.05. The ns stands for no significance.Error bars are ±SE.

FIG. 9 (FIG. 9 A to D) shows the content of BXDs compounds DIMBOA-Glc(A), DIMBOA (B), HMBOA-Glc (C), and HDMBOA-Glc (D) in second leaves ofHtn1 NILs and mutants at 21 days after sowing. Further information isprovided in Example 12 below. The statistics were conducted usingStudent's t test (P=0.05) in eight biological replicates. The asterisksrepresent a significant difference of **p<0.01 or *p<0.05. The ns standsfor no significance. Error bars are ±SE.

FIG. 10 shows relative RLK1 expression in tissues. These samples wereharvested from 21 days old seedlings without pathogen inoculation.Different lower case letters in the graphs indicate a difference whichis statistically different. See also Example 13 below.

FIG. 11 (FIGS. 11 A and P) shows the transcription levels of genes inHtn1-NILs and the corresponding parental lines. The expression of genes(FIG. 11 A) ZmWAK-RLK1, (FIG. 11 B) Bx1, (FIG. 11 C) Igl, (FIG. 11 D)Bx2, (FIG. 11 E) Bx3, (FIG. 11 F) Bx4, (FIG. 11 G) Bx5, (FIG. 11 H) Bx6,(FIG. 11 I) Bx7, (FIG. 11 J) Bx8, (FIG. 11 K) Bx9, (FIG. 11 L) Bx10/11,(FIG. 11 M) Bx12, (FIG. 11 N) Bx13, (FIG. 11 O) Glu1 and (FIG. 11 P)Glu2 were quantified. The different colors and patterns of the barsindicate timepoints before and after infection as shown in the legendfor FIG. 11 A which also applies for FIG. 11 B to P. The statistics wereconducted separately in w22 and B37 genetic background using Tukey's HSD(P=0.05) in four biological replicates. Error bars are ±SE. Differentlower case letters in the graphs indicate a difference which isstatistically different. See also Example 10 below.

FIG. 12 (FIG. 12 A to C) shows ZmWAK-RLK1 localization to the plasmamembrane. (FIG. 12 A-B) Fluorescent signals in onion epidermal cellsafter transient expression of ZmWAK-RKL1-eGFP (SEQ ID NO: 9) and thepositive control PIP2A-mCherry that is known to localize to the plasmamembrane (Kammerloher, Werner, et al. “Water channels in the plantplasma membrane cloned by immunoselection from a mammalian expressionsystem.” The Plant Journal 6.2 (1994): 187-199). Signals are shownbefore (FIG. 12 A) and after (FIG. 12 B) plasmolysis with 0.8 Mmannitol. (FIG. 12 C) Fluorescent signals in N. benthamiana leaves twodays after infiltration. Notably, the signals shown in white in therespective graphs correspond to the originally green (column 1RLK_eGFP), red (column 2 PIP2A_mCherry), and yellow (merge, column 4)fluorescent signal. Column 3 (DIC) represents the differentialinterference contrast to visualize the cellular structures as control.

FIG. 13 is a table showing the log FC and annotation of 215 thedifferentially expressed genes (DEGs) detected in B37Htn1/B37 andw22Htn1/w22 in at least one of timepoints.

DETAILED DESCRIPTION

Based on the experiments and data underlying the present invention, itwas found that the genes involved in the biosynthesis of benzoxazinoids(BXDs) and derivatives thereof, mainly hydrolysis based derivatives likehydroxamic acids, are not only involved in the defense mechanism againstE. turcicum but rather can also effect and mediate plant-defense, i.e.,resistance mechanisms against various other fungal pathogens in a seriesof crop plants. The present invention thus implements both the linkbetween a wall associated kinases (WAK), downstream signaling molecules,such as Bx1, Bx2, Bx6, Bx14 and Igl, or any other enzyme involved in thebenzoxazinoid synthesis pathway, and the decrease of BXD secondarymetabolites and further technically implements the finding that thisdecrease of BXD secondary metabolites is associated with increasedfungal resistance.

The present invention thus provides in a first aspect a method forproducing a plant having increased fungal resistance, wherein the fungalresistance is regulated by at least one wall-associated kinase, themethod comprising: (i) (a) providing at least one plant cell, tissue,organ, or whole plant having a specific genotype with respect to thepresence of at least one gene encoding a wall-associated kinase in thegenome of said plant cell, tissue, organ, or whole plant; or (i) (b)introducing at least one gene encoding at least one wall-associatedkinase into the genome of at least one cell of at least one of a plantcell, tissue, organ, or whole plant; and (ii) (a) modifying at least onegene encoding at least one wall-associated kinase in the at least oneplant cell, tissue, organ, or whole plant; and/or (ii) (b) modulatingthe expression level of at least one wall-associated kinase and/or thetranscription level, the expression level, or the function of at leastone molecule within the signaling pathway from the at least onewall-associated kinase to the synthesis of at least one benzoxazinoid orwithin the synthesis pathway of at least one benzoxazinoid in the atleast one plant cell, tissue, organ, or whole plant; and (iii) producinga population of plants from the at least one plant cell, tissue, organ,or whole plant; and (iv) selecting a plant having an increased fungalresistance, from the plant population, based on the determination of areduced synthesis of a benzoxazinoid preferably in response to a fungalinfection, wherein the selected plant have an increased fungalresistance based on the reduced synthesis of a benzoxazinoid, and/orwherein the synthesis of the benzoxazinoid is regulated by the at leastone wall-associated kinase.

Wall associated kinases (WAKs) have recently been identified as majorcomponents of fungal and bacterial disease resistance in several cerealcrop species. However, the molecular mechanisms of WAK-mediatedresistance are presently largely unknown. According to this invention,the function of the maize gene ZmWAK-RLK1 (Htn1) that confersquantitative resistance to northern corn leaf blight (NCLB) caused bythe hemibiotrophic fungal pathogen Exserohilum turcicum wasinvestigated. ZmWAK-RLK1 (Htn1) was found to localize to the plasmamembrane and its presence resulted in a modification of the infectionprocess by specifically reducing pathogen penetration into host tissues.Furthermore, the ubiquitous expression of ZmWAK-RLK1 and the findings onthe signaling pathway downstream of ZmWAK-RLK1 demonstrate the functionof this and associated wall-associated kinases as master regulators andcrucial signaling mediators in plant defense against fungal disease.

A transcriptome analysis of near-isogenic lines (NILs) differing forZmWAK-RLK1 revealed that several genes involved in the biosynthesis ofthe secondary metabolites benzoxazinoids (BXDs) were differentiallyexpressed in the presence of ZmWAK-RLK1. Particularly the content of BXDcompounds DIMBOA-Glc, DIMBOA, HMBOA-Glc and DIM₂BOA-Glc weresignificantly lower in the NILs with ZmWAK-RLK1. Furthermore,DIM₂BOA-Glc, which is an inactive glucoside of BXDs, was significantlyelevated in ZmWAK-RLK1 mutants with compromised NCLB resistance. Inaddition, maize mutants that were affected in BXDs biosynthesis showedreduced susceptibility to E. turcicum infection at the seedling stage.We thus conclude that BXD biosynthesis increases susceptibility to E.turcicum infection and that the ZmWAK-RLK1-mediated NCLB resistanceresults from a reduction of these compounds. These findings indicate anovel link between WAKs underlying quantitative disease resistance andthe defense mechanism mediated by the secondary metabolites BXDs thathave been known for their involvement in cereal insect resistance. Theterm “WAK” as used herein may comprise a plant receptor-like kinaseassociated with the signal transduction directly or indirectly effectingthe biosynthesis of genes involved in the BXD synthesis, or interactingwith signaling mechanism and/or protein-protein interactions beinginvolved in the BXD synthesis.

The plant immune response “caused” by a WAK can thus be of direct orindirect nature. As it is known to the skilled person, receptor-likekinases usually comprise at least one extracellular signaling domain,e.g., for sensing PAMPs and/or DAMPs, a transmembrane domain, and anintracellular kinase domain. The kinase domain allows the WAK totransform the extracellular signal into an intracellular responsetransferred via a cascade of proteins involved in the downstream signaltransduction. Usually, receptor kinases thus indirectly initiate theactivation and transfer into the nucleus/organelle of a transcriptionfactor which regulates the transcription of a target gene. Furthermore,plant WAKs can trigger defense responses such as reactive oxygen species(ROS) accumulation through the activation of a NADPH oxidase, nitricoxide production, callose deposition, besides a MAP kinase-mediatedactivation of defense gene expression. The terms “causing” or “caused”as used herein in the context of a WAK or another plant receptor kinaseis thus to be construed broadly to comprise any direct or indirecteffect the activity of the WAK can have on downstream signalingmolecules, wherein the molecules can be selected from at least one aminoacid sequence, preferably an enzyme in the signal transduction cascadedownstream of the WAK or a peptide being able to stimulate or inhibitcomplex formation downstream of the WAK or signal transductiondownstream of a WAK, a metabolite, such as any secondary metaboliteproduced by a plant, a ROS, or an indirect effect on the regulation ofthe transcription and/or translation of another downstream gene/protein.A physical interaction of the WAK in the form of a signaling complex mayoccur to cause an action. In another embodiment, the action caused bythe WAK is mediated by a downstream molecule, e.g., a downstream kinasephosphorylating another molecule, in an indirect way. In the terminalpart of the WAK signaling cascade, a transcription activator orrepressor can be induced to regulate the transcription of a target geneof a WAK, preferably a target gene in the jasmonic acid and/or BXDbiosynthesis pathway. Besides a protein-DNA interaction, WAK signalingcan also imply protein-protein interactions influencing the BXDbiosynthesis pathway.

In another aspect, the methods of the present invention further maycomprise the step of introducing, modifying and/or modulating at leastone further or other gene into at least one plant cell, tissue, organ,or whole plant to provide a synergistic effect in increasing fungaldisease by decreasing the synthesis of at least one BXD compoundassociated with fungal resistance. In preferred embodiments, the atleast one further or other gene is selected from a bx1, bx2, igl, bx6,bx11, bx14, opr2, lox3 or aoc1 gene (SEQ ID NOs: 10, 12, 14, 16, 18, 20,22, 24 or 26, respectively), or a homologous gene thereof, or therespective proteins encoded by said genes as set forth exemplary in SEQID NOs: 11, 13, 15, 17, 19, 21, 23, 25 or 27, respectively, or homologsthereof. As disclosed herein, certain genes involved in the jasmonicacid pathway, the ethylene pathway, the lignin synthesis pathway, aplant defense pathway, a further receptor-like kinase pathway, or a cellwall pathway, and preferably certain genes involved in the jasmonic acidpathway, contribute to the signaling pathway of at least one functionalWAK, wherein there may be a synergistic effect provided by the presenceof a specific functional WAK and a specific non-functional or lessfunctional gene of the jasmonic acid pathway, as the presence of bothwill contribute to an even significantly reduced amount of a BXDcompound of interest and thus a more than additive increase in fungalresistance.

The present invention thus provides specific target genes which can bemodulated in addition or alternatively to the at least one WAK ofinterest to provide a significantly improved fungal defense strategy fora plant of interest. These results are based on different functionalstudies including comparative transcriptome analysis in defined specificWAK genotypes, namely in two pairs of near isogenic lines, w22 andW22Htn1 as well as B37 and B37Htn1 (see Example 10 below), after fungalspecific stimuli by analyzing the RNA sequencing datasets. Furthermore,additional RT-qPCR experiments and systematic RNA sequencing wereconducted to decipher the plant immune network as triggered by a WAK,e.g., Htn1, (Examples 6 and 10 and Tables 1 and 2). These datademonstrated a cross-talk between the WAK and the benzoxazinoidsynthesis and jasmonic acid pathway and thus provided new candidates toprovide new elite plant lines comprising both a specific WAK as well asa specific genotype with respect to enzymes involved in thebenzoxazinoid synthesis and jasmonic acid pathway.

In one embodiment according to the present invention, the method forproducing a plant having increased fungal resistance may comprise themodification of at least one gene encoding at least one wall-associatedkinase, and optionally at least one further or other gene, for example abx1, bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, in the at leastone plant cell, tissue, organ, or whole plant. The modification can beconducted by any means of plant breeding, including classical and modernmethods of plant breeding, and/or techniques of molecular biology.Classical plant breeding methods may comprise the deliberateinterbreeding (crossing) of closely or distantly related species toproduce new crops with desirable properties. Plants are crossed tointroduce traits/genes from a particular variety into a new geneticbackground to provide plants having modified and/or increased quality,yield, tolerance (against abiotic stress), resistance (against bioticstress), etc., characteristics. Breeding nowadays also includes methodslike marker-assisted selection, reverse breeding and the targetedcombination with molecular biology tools known and available to theskilled person.

In one embodiment according to the present invention, the modulation ormodulating of at least one wall-associated kinase, and/or of at leastone further or other gene, for example a bx1, bx2, igl, bx6, bx11, bx14,opr2, lox3 or aoc1 gene, can thus comprise at least one of modulatingthe expression level of at least one wall-associated kinase, preferablyincreasing the expression at least one wall-associated kinase, and/ormodulating the function or activity of and/or activity of at least onewall-associated kinase, for example, by providing at least one moleculeinteracting with the extracellular signalling domain of at least oneWAK, e.g., an activator, or by providing at least one moleculeinteracting with the intracellular signalling domain of at least oneWAK, such as a molecule inducing or inhibiting kinase activity. In oneembodiment, the modulation or modulating of at least one wall-associatedkinase, and/or of at least one further or other gene, for example a bx1,bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, can comprise thetargeted introduction of at least one mutation into a WAK and/or afurther or other gene of interest to modulate the activity of the WAKand the further or other protein encoded by the at least one further orother gene in a targeted way. Embodiments comprising the modulation ofat least one wall-associated kinase thus aim at influencing the activityof the at least one WAK within without modifying the nucleic acidsequence and thus possibly the amino acid sequence of a WAK of interest.In certain embodiments, wherein at least one further or other gene, forexample a bx1, bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, ismodulated, the modulation may aim at reducing the activity of a at leastone allele of a bx1, bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 geneto decrease the amount of BXD compound synthesized. For example, BX1 andIGL enzymes are accountable for the bulk of BX biosynthesis. Therefore,inhibiting the presence of a functional BX enzyme, preferably a BX1, BX2or BX6 enzyme, or an Igl enzyme can contribute to the provision of areduced BXD synthesis and thus an increased fungal resistance in a plantof interest.

The modulation according to the present invention can comprise anydirect or indirect interaction between two molecules, i.e., areceptor-ligand interaction, a transcription factor-transcription factorbinding site interaction, an interaction of an enzyme, e.g., a kinasewith its target site, an interaction of a peptide or nucleic acidmodulator with a target site, an antibody-antigen interaction, aninteraction with a DNA or histone binding protein and its cognate ligand(DNA or histone), a hybridization between two nucleic acidsequences/molecules and the like.

In one embodiment, the transcription level of at least one WAK within atleast one cell of at least one of a plant cell, tissue, organ, or wholeplant can be modified or modulated by specifically influencing aregulatory sequence of a WAK gene. In another embodiment, the modulationaffects at least one gene, for example a bx1, bx2, igl, bx6, bx11, bx14,opr2, lox3 or aoc1 gene. This modulation or modification can comprisethe introduction of at least one specific mutation, for example toactivate a promoter of interest, or the modulation or modification canbe in trans by providing a transcription factor modulating thetranscription of at least one WAK gene, wherein the at least one WAKgene according to all embodiments of the present invention may comprisean endogenously occurring WAK gene, or a WAK gene introduced into atleast one cell of at least one of a plant cell, tissue, organ, or wholeplant.

According to the various aspects and embodiments of the presentinvention, a signalling pathway from the at least one wall-associatedkinase to the synthesis of at least one benzoxazinoid in at least oneplant cell, tissue, organ, or whole plant thus implies the whole chainof molecular actions downstream of a WAK as sensing molecule triggeringa signalling cascade involving various different effectors until thesynthesis of a BXD compound.

In one embodiment according to the various aspects of the presentinvention, the at least one wall-associated kinase is WAK-RLK1,preferably selected from Htn1, Ht2, or Ht3, or an allelic variantthereof, a mutant or a functional fragment thereof, or a gene encodingthe same, preferably wherein the at least one wall-associated kinase a)is encoded by a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO: 1 or 7, or a functional fragment thereof, b) is encoded bya nucleic acid molecule comprising the nucleotide sequence having atleast 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to nucleotide sequence of SEQ ID NO: 1 or 7, preferably overthe entire length of the sequence, c) is encoded a nucleic acid moleculehybridizing with a complementary sequence to a) or b) under stringentconditions, d) is encoded by a nucleic acid molecule comprising thenucleotide sequence coding for an amino acid sequence of SEQ ID NO: 2 or8, or a functional fragment thereof, e) is encoded by a nucleic acidmolecule comprising the nucleotide sequence coding for an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8,preferably over the entire length of the sequence, f) comprising theamino acid sequence of SEQ ID NO: 2 or 8, or g) comprising an amino acidsequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% sequence identity to amino acid sequence of SEQ ID NO: 2 or 8,preferably over the entire length of the sequence, provided that anysequence of a) to g), optionally after expression, still encodes atleast one functional Htn1, Ht2, or Ht3, or an allelic variant, a mutant,or a functional fragment thereof. In a preferred embodiment, the atleast one wall-associated kinase is selected from Htn1 (RLK1) or anallelic variant, a mutant or a functional fragment thereof, or a geneencoding the same. Variants may further comprise any functional splicevariant of a WAK gene. As it is known to the skilled person, eukaryoticmRNA comprising introns is spliced during processing from a precursormRNA into a mature mRNA giving rise to a protein after translation(protein biosynthesis).

“Functional” or “functional fragment” or “variant” as used in connectionwith a WAK or any other receptor-like kinase or any at least one furthergene/protein, for example a bx1, bx2, igl, bx6, bx11, bx14, opr2, lox3or aoc1 gene, or the corresponding proteins Bx1, Bx2, Igl, Bx6, Bx11,Bx14, Opr2, Lox3 or Aoc1, according to the present disclosure means afragment of an amino acid or nucleic acid sequence with reference to therespective (longer) sequence occurring in the natural environment of aplant genome of interest, whereas the functional fragment stillcomprises—optionally after transcription, processing and translation—atleast one function of the respective parent sequence. The functionalfragment may be less sterically demanding and thus more convenient forcertain approaches. Furthermore, the functional fragment may be fused toanother domain to create a fusion molecule for functional assays, e.g.,a fusion with a gene encoding a protein having fluorescence activity. Inanother embodiment, the functional fragment may be fused to a tag andthe like. Therefore, a functional fragment may also comprise a sequencecomprising codon optimizations on the nucleic acid level, or comprisingcertain mutations, said mutations not influencing the activity orfunction of a WAK, or another receptor-like kinase of interest.

Preferably, any function variant at least comprises a truncated form ofthe extracellular signalling domain of a WAK and an active intracellularkinase domain, wherein the intracellular kinase domain is able toinitiate downstream signalling. Notably, the extracellular domain, thetransmembrane domain and/or the intracellular kinase domain of a WAKaccording to the present invention can comprise at least one mutation.Said mutation may lead to an increased signalling activity to representa functional variation or functional mutation in the sense of thepresent invention.

In certain aspects according to the present invention, at least onefurther or other gene, for example a bx1, bx2, igl, bx6, bx11, bx14,opr2, lox3 or aoc1 gene, is introduced, and/or modified and/or modulatedaccording to the methods of the present invention, variants or mutantsrepresenting “loss-of-function”, or having reduced activity might bespecifically preferred for the purpose of the present invention in casethat the at least one variant or mutant results in a decreased BXDsynthesis. Particularly, it was found according to the present inventionthat there is a cross-talk between the WAK signaling pathway and the BXDsynthesis pathway, mainly the BXD synthesis pathway as mediated by Bx1,Bx2, Bx6, Bx11 and BX14 and/or Igl, wherein the targeted insertion,modulation or modification of at least one WAK, or the gene encoding thesame, and a further effector, or the gene encoding the same, for examplea bx1, bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 gene, contribute toa enhanced fungal resistance, in particular NCLB resistance, in a plantas the regulation of both pathways leads to a reduced BXD signature.Wherein the WAK pathway plays the role of the general “pacemaker” inthis regulatory network which senses and forwards signals due to itsrecognition and kinase function, there is also a feedback regulationbetween the further effectors involved in the jasmonic acid and BXDsynthesis pathway. The master regulator function of WAK is demonstratedby the fact that the combined expression of Bx1 and Igl was consistentlylower in genotypes with ZmWAK-RLK1 (FIGS. 11 B and C and Example 10)demonstrating that ZmWAK-RLK1 and other WAKs have the capacity to inducea concerted action also regulating the benzoxazinoid pathway and thejasmonic acid pathway.

In one embodiment according to the various aspects of the presentinvention, the method for producing a plant having increased fungalresistance thus comprises the introduction and/or modification and/ormodulation of at least one WAK, or a gene encoding the same, wherein theWAK at least comprises a functional intracellular kinase domain, forexample a sequence selected from SEQ ID NOs: 1, 2, 7, or 8, or anallelic variant or mutant thereof, and wherein the method furthercomprises the introduction and/or modification and/or modulation of atleast one BX or Igl protein, or a gene encoding the same, wherein thecorresponding bx gene, or the Igl gene comprises at least one mutation,or wherein the bx gene, or the Igl gene is of a specific genotype isknocked-out, so that the WAK activity and the decreased or deleted BXprotein or Igl protein activity results in a decreased BXD biosynthesis.

Receptor-like kinases can be further divided into RD and non-RD kinases,depending on the presence or absence of an arginine residue at thecatalytic site of the kinase domain. ZmWAK-RLK2 contains an RD kinaseand ZmWAK-RLK1 has a non-RD kinase domain (cf. for instance positions505 and 506 of SEQ ID NO: 2, amino acids F (phenylalanine) and D(aspartic acid), respectively). Most receptor-like kinases involved inplant immunity identified so far belong to the non-RD kinases, whereasRD kinases are thought to play a role in other processes such asdevelopment. Variants of SEQ ID NO: 2 have been constructed (cf. SEQ IDNOs: 3 to 6 and Hurni et al., 2015). It was found that mutations atpositions M455, G497 and G548 (with reference to SEQ ID NO: 2) mayresult in a higher susceptibility to NCLB. All said positions reside inthe serine threonine kinase domain of ZmWAK-RLK1. A functional variantaccording to the present invention will thus avoid any mutation orcombination of mutations in the kinase domain of a WAK which results indecreased fungal resistance. Exemplary mutants of SEQ ID NO: 2 arepresented with SEQ ID NOs: 3 and 4 (RLK1b, M455I) and SEQ ID NOs: 5 and6 (RLK1d, G497E). A further mutant analyzed herein, RLK1f, comprises amutation G548R in comparison to the wild-type sequence according to SEQID NO: 2. All mutants were tested in comparison to the respective sisterlines as described herein. Based on these structural data the importanceof a functional intracellular kinase domain of a WAK could be deduced.Therefore, a functional variant or a functional mutant of a WAK maycomprise at least one mutation in comparison to the cognate wild-typesequence which at least one mutation does not disturb the downstreamsignaling of the WAK in that sense that a functional mutant or variantwill decrease the level of a specific BXD compound to in turn increasefungal resistance of a plant, plant cell, tissue, or organ comprisingsuch a functional variant of a WAK, or the sequence encoding the same.

According to certain embodiments of the present invention, more than onegene encoding a WAK, or a functional fragment thereof, or the sequenceencoding the same, can be introduced into, or modulated or modified inat least one plant cell, tissue, organ, or whole plant. Theintrogression of several WAKs can have a synergistic effect in providingenhanced fungal resistance, particularly in case an elite line can beestablished based on the staggering of more than one WAK into the genomeof a plant of interest according to the disclosure of the presentinvention. As described herein, WAKs represent the key signallingmolecules initiating an immune cascade downstream of and mediated by theintracellular kinase domain of the WAKs. Therefore, more than one WAKmay thus have a dosage effect positively downregulating BXD synthesisand thus increasing fungal resistance in a plant, in particular a cropplant, of interest. Furthermore, at least one further gene or protein,preferably being selected from any one of SEQ ID NOs: 10 to 27 orhomologous genes or homologs thereof, can be additionally oralternatively modified as detailed above to provide a plant cell,tissue, organ or whole plant as material for producing a plant withimproved fungal resistance properties, preferably resistance againstNCLB. Further target sequences to be modified having an implication inthe cross-talk between WAK signalling and BXD biosynthesis are disclosedin Tables 1 and 3 herein.

In a further embodiment according to the present invention, there isprovided a method, wherein the reduced synthesis of at least onebenzoxazinoid is achieved by providing at least one wall-associatedkinase, an allelic variant, a mutant or a functional fragment thereof,or a gene encoding the same, wherein the at least one wall-associatedkinase comprises a sequence which can directly or indirectly influencethe benzoxazinoid pathway and at least one further plant metabolicpathway, preferably a disease resistance associated pathway, wherein theplant metabolic pathway is selected from the group consisting of thejasmonic acid pathway, the ethylene pathway, the lignin synthesispathway, a defense pathway, a receptor-like kinase pathway, a cell wallassociated pathway, preferably, wherein the at least one further plantmetabolic pathway is the jasmonic acid pathway and wherein the reducedsynthesis of at least one benzoxazinoid is achieved by an decreased ordown-regulated Igl and/or Bx1 expression as induced by at least one WAKof interest.

Several differentially expressed genes (DEGs) identified by theinventors of the present invention belonged to several different immunenetworks and to different disease resistance associated pathwaysincluding benzoxazinoids (BXDs) biosynthesis, (phytohormone) jasmonicacids (JAs), ethylene, lignin, defense and receptor-like kinases as wellas cell wall were found in Htn1 NILs (Example 10, FIG. 13).Surprisingly, six genes of the BXDs biosynthesis pathway showeddifferential expression in at least one timepoint, including Bx1 (SEQ IDNOs: 10 and 11), Bx2 (SEQ ID NOs: 12 and 13), Igl-like (SEQ ID NOs: 14and 15), Bx6 (SEQ ID NOs: 16 and 17), Bx11 (SEQ ID NOs: 18 and 19) andBx14 (SEQ ID NOs: 20 and 21) (see also FIG. 13, column 2 of table forfurther reference to publicly available data base entries for gene IDsand names) demonstrating a cross-regulatory network between the WAK andfurther pathways in fungal defense. This was particularly surprisingbecause BXDs as secondary metabolites have so far not been associatedwith defense against fungi mediated by WAK kinases. More surprisingly,the transcriptome data and functional assays also, for the first time,revealed DEGs that are part of immune networks including thephytohormone jasmonic acids that plays a central role in regulatingresistance against hemibiotrophic and necrotrophic diseases. JAstreatment can induce the accumulation of BXD compounds (Oikawa, Akira,Atsushi Ishihara, and Hajime Iwamura. “Induction of HDMBOA-Glcaccumulation and DIMBOA-Glc 4-O-methyltransferase by jasmonic acid inpoaceous plants.” Phytochemistry 61.3 (2002): 331-337; Oikawa, Akira, etal. “Accumulation of HDMBOA-Glc is induced by biotic stresses prior tothe release of MBOA in maize leaves.” Phytochemistry 65.22 (2004):2995-3001). The present invention thus provides evidence that there isan additional link between the WAK kinase signaling pathway and thejasmonic acid pathway which paves the way for a variety of new diseaseand particularly fungal resistance strategies as disclosed herein. Theenzymatic properties of IGL are similar to BX1, but the transcriptionalregulation of their corresponding genes is different. Like other Bxgenes, Bx1 is constitutively expressed during the early developmentalstages of the plant, which correlates with endogenous BX levels. Plantscarrying the mutant alleles of the Bx1 gene produce only a fraction ofthe BXs that are found in Bx1 wild-type plants. Therefore, a WAKaccording to the present invention may act as a master regulatorbridging anti-fungal signalling with the effectors of the jasmonic acidpathway and other pathways, preferably an effector selected from thegroup consisting of SEQ ID NOs: 10 to 27 or homologous genes or homologsthereof.

In one embodiment, the introduction at least one additional geneencoding at least one wall-associated kinase into at least one cell ofat least one of a plant cell, tissue, organ, or whole plant may comprisethe introduction of a nucleic acid sequence, comprising DNA and/or RNAin a single stranded and/or double stranded form, or an amino acidsequence, by means of breeding techniques, or by means of molecularbiology to transfer a functional WAK of interest, or an additionalfunctional WAK of interest, or the sequence encoding the same, into atleast one cell of interest. Said at least one additional gene can bealso any gene, wherein the resulting protein/enzyme is involved in theBXD biosynthesis pathway or in a jasmonic acid pathway, such as Bx1,Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Igl,Glu1, Glu2, OPR2, LOX3 or AOC1 (see FIG. 13 and SEQ ID NOs: 10 to 27),or any variant thereof, or a combination of the aforementionedgenes/proteins. Preferably, the at least one additional gene comprisesat least one mutation which changes the function of the naturallyoccurring respective additional gene, wherein the mutation, in thecoding or within a regulatory region, causes decreased synthesis of therespective BXD compound, or wherein the mutation, in a regulatoryregion, such as a promoter region, or in a coding region, causes areduced signal transduction from a WAK kinase located upstream in thesignalling cascade so the said mutation results in a decreased synthesisof a BXD compound. In another embodiment, the gene encoding at least oneof Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14,Igl, Glu1, Glu2, OPR2, LOX3 or AOC1 may be deleted or partially deletedwithin the genome of a plant cell of interest, or the gene may bemodified in a targeted way.

Further enzymes involved in the regulation of the BXD synthesis whichcan be modulated, introduced or modified according to the methods of thepresent invention to achieve an increased fungal resistance in a plantcell, plant or plant material pathway are selected from the group ofjasmonic synthesis pathway enzymes, including 12-oxo-phytodienoic acidreductase 2 (OPR2), Lipoxygenase 3 (LOX3) or Allene oxide cyclase 1(AOC1), ethylene pathway enzymes, such as S-adenosylmethionine synthase,lignin pathway enzymes, such as, for example, Caffeoyl-CoAO-methyltransferase 1 (OMT1) or OMT2, enzymes and proteins involved inplant defense mechanisms, such as, for example SAF1—Safener induced 1;Glu2athione S-transferase, and any combination thereof.

Presently, WAKs are the only known proteins that can physically link thecell wall to the plasma membrane (Brutus, Alexandre, et al. “A domainswap approach reveals a role of the plant wall-associated kinase 1(WAK1) as a receptor of oligogalacturonides.” Proceedings of theNational Academy of Sciences 107.20 (2010): 9452-9457). Therefore,further structurally and functionally related cell wall spanning orassociated kinases are suitable as WAKs according to the presentinvention, e.g., maize qHSR1 (Zuo, Weiliang, et al. “A maizewall-associated kinase confers quantitative resistance to head smut.”Nature genetics 47.2 (2015): 151-157), or rice OsWAK/Xa4 gene conferringquantitative rice blight resistance by strengthening the cell wall (Huet al. 2017).

In another embodiment according to the present invention, the step ofintroducing at least one gene into at least one cell of at least one ofa plant cell, tissue, organ, or whole plant may comprise theintroduction of a gene, wherein the amino acid sequence or enzymeencoded by said gene is involved in the catalytic pathway downstream ofa WAK kinase, wherein the additional gene is introduced alone, ortogether with at least one gene encoding a WAK kinase or a variantthereof.

“Benzoxazinoids” or “BXDs” are a class of indole-derived plant chemicaldefenses comprising compounds with a2-hydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton and their derivatives.BXDs have been described as phytochemicals in monocots, includinggrasses, including important cereal crops such as maize, wheat and rye,as well as a certain dicot species. The term “BXDs” as used hereinrefers to both benzoxazinones (glucosides and corresponding agluconescontaining a 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton) and theirdownstream derivative products during metabolic pathways,benzoxazolinones, as well as any intermediates. The term BXD may thusalso comprise a derivative being the result of the activity ofhydrolyzing glucosidases found in plastids, cytoplasm, and cell walls,or derivatives and intermediated being the result of degradation tobenzoxazolinones via oxo-cyclo/ring-chain tautomerism. Further comprisedare downstream metabolites directly being derivable from anybenzoxazolinone. The term “BXDs” shall further comprise any open form,nitrenium form or complex, e.g., a metal complex from a BXD. BXD basicstructures are represented in FIG. 1.

By the term “reduced/decreased synthesis of a benzoxazinoid” or “reducedsynthesis of at least one benzoxazinoid” or “reducing the amount of atleast one benzoxazinoid” or “reduction on BXDs content” or “reducedamount of a BXD compound” or the like, is meant that the plant cell,tissue, organ, or whole plant according to the present invention exhibitan amount of a benzoxazinoid, at least one benzoxazinoid or thebenzoxazinoid of interest which is reduced by at least 10%, 15%, 20% or25%, preferably by at least 30%, 35%, 40% or 45%, more preferably by atleast 50%, 60% or 70% as compared to a corresponding control plant cell,control tissue, control organ, or control whole plant of the samegenotype, but lacking the modification of the at least one gene encodingat least one wall-associated kinase or the modulation of the expressionlevel of at least one wall-associated kinase and/or the transcriptionlevel, the expression level, or the function of at least one moleculewithin the signaling pathway from the at least one wall-associatedkinase to the synthesis of at least one benzoxazinoid or within thesynthesis pathway of at least one benzoxazinoid. In one embodimentaccording to the various aspects of the present invention, thebenzoxazinoid whose synthesis is regulated by the at least onewall-associated kinase and optionally regulated by the at least onefurther enzyme of the jasmonic acid and/or benzoxazionoid pathway isselected from at least one of DIM₂BOA, DIMBOA, HMBOA, HM₂BOA, HDMBOA,HDM₂BOA, HBOA, DHBOA, DIBOA or TRIBOA, the aforementioned benzoxazinoidbeing in the glucoside or aglucone form, or a benzoxazolinone, or anycombination of the aforementioned benzoxazinoids, preferably wherein thebenzoxazinoid whose synthesis is regulated by the at least onewall-associated kinase is selected from at least one of DIM₂BOA, DIMBOA,HMBOA or HDMBOA, the aforementioned benzoxazinoid being in the glucosideor aglucone form, or any combination of the aforementionedbenzoxazinoids.

In one embodiment according to the various aspects of the presentinvention, a reduced content of BXDs can be achieved by introducing atleast one gene encoding at least one wall-associated kinase into atleast one cell of at least one of a plant cell, tissue, organ, or wholeplant, wherein the at least one wall-associated kinase causes a reducedsynthesis of at least one BXD. More than one WAK encoding gene anddifferent allelic variants of a WAK gene may be introduced into a cellof interest in addition to a WAK gene potentially already being presentin the genome of a plant cell of interest. The presence of several WAKsor receptor-like kinases involved in the BXD synthesis may thus befavourable in order to increase the copy number and thus the dosageeffect of a gene of interest.

In one embodiment, quantitative NCLB disease resistance is based on adecrease of the biosynthesis of at least one secondary metabolite BXDs,preferably DIM₂BOA-Glc, DIMBOA, HMBOA, DIMBOA-Glc or HMBOA-Glc, and themethods according to the various aspects of the present inventioncomprise the addition of a scavenger molecules interacting with and thisneutralizing the activity of at least one secondary metabolite BXD toreduce the amount of the of at least one secondary metabolite BXDsusceptibility component to decrease fungal infection at least one plantcell, tissue, organ, or whole plant.

According to the present invention, there are thus provided methods forproducing a plant having increased fungal resistance, wherein the fungalresistance is regulated by at least one wall-associated kinase.“Regulated” in this context thus implies a direct or indirect regulationmediated by at least one wall-associated kinase. This regulation mayimply a signalling cascade initiated by the at least one wall-associatedkinase and proceeding through further molecules involved in thesignalling cascade. The regulation can be on a protein, RNA or nucleicacid level. Furthermore, the regulation may imply a cross-talk orfeedback regulation, for example implying a Bx1, Bx2, Bx3, Bx4, Bx5,Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Igl, Glu1, Glu2, OPR2, LOX3or AOC1 enzyme, or the gene encoding the same, or the transcriptionalregulation of such a further gene encoding Bx1, Bx2, Bx3, Bx4, Bx5, Bx7,Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Igl, Glu1, Glu2, OPR2, LOX3 orAOC1, or a modulation or modification of a gene encoding Bx1, Bx2, Bx3,Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Igl, Glu1, Glu2,OPR2, LOX3 or AOC1.

In one embodiment, the pathogen according to the present disclosure is afungal pathogen infesting a plant. The disease caused by a fungalpathogen and the respective fungus may be selected from Plume blotchSeptoria (Stagonospora) nodorum, Leaf blotch (Septoria tritici), Earfusarioses (Fusarium spp.), Late blight (Phytophthora infestans),Anthrocnose leaf blight or Anthracnose stalk rot (Colletotrichumgraminicola (teleomorph: Glomerella graminicola Politis) Glomerellatucumanensis), Curvularia leaf spot (Curvularia clavata, C.eragrostidis, =C. maculans (teleomorph: Cochliobolus eragrostidis),Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolusintermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus),Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvulariasenegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus),Didymella leaf spot (Didymella exitalis), Diplodia leaf spot or streak(Stenocarpella macrospora=Diplodialeaf macrospora), Brown stripe downymildew (Sclerophthora rayssiae var. zeae), Crazy top downy mildew(Sclerophthora macrospora=Sclerospora macrospora), Green ear downymildew (Sclerospora graminicola), Leaf spots (various minor leaf spots)(Alternaria alternata, Ascochyta maydis, A. tritici, A. zeicola,Bipolaris victoriae=Helminthosporium victoriae (teleomorph: Cochliobolusvictoriae), C. sativus (anamorph: Bipolaris sorokiniana=H.sorokinianum=H. sativum), Epicoccum nigrum, Exserohilumprolatum=Drechslera prolata (teleomorph: Setosphaeria prolata) Graphiumpenicillioides, Leptosphaeria maydis, Leptothyrium zeae, Ophiosphaerellaherpotricha, (anamorph: Scolecosporiella sp.), Paraphaeosphaeriamichotii, Phoma sp., Septoria zeae, S. zeicola, S. zeina, Northern cornleaf blight (Setosphaeria turcica (anamorph: Exserohilumturcicum=Helminthosporium turcicum), Northern corn leaf spot(Cochliobolus carbonum (anamorph: Bipolaris zeicola=Helminthosporiumcarbonum)), Phaeosphaeria leaf spot (Phaeosphaeria maydis=Sphaerulinamaydis), Rostratum leaf spot (Setosphaeria rostrata, (anamorph:Helminthosporium rostratum)), Java downy mildew (Peronosclerosporamaydis=Sclerospora maydis), Philippine downy mildew (Peronosclerosporaphilippinensis=Sclerospora philippinensis), Sorghum downy mildew(Peronosclerospora sorghi=Sclerospora sorghi), Spontaneum downy mildew(Peronosclerospora spontanea=Sclerospora spontanea), Sugarcane downymildew (Peronosclerospora sacchari=Sclerospora sacchari), Sclerotium earrot (southern blight) (Sclerotium rolfsii Sacc. (teleomorph: Atheliarolfsii)), Seed rot-seedling blight (Bipolaris sorokiniana, B.zeicola=Helminthosporium carbonum, Diplodia maydis, Exserohilumpedicillatum, Exserohilum turcicum=Helminthosporium turcicum, Fusariumavenaceum, F. culmorum, F. moniliforme, Gibberella zeae (anamorph: F.graminearum), Macrophomina phaseolina, Penicillium spp., Phomopsis sp.,Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicariasp.), Selenophoma leaf spot (Selenophoma sp.), Yellow leaf blight(Ascochyta ischaemi, Phyllosticta maydis (teleomorph: Mycosphaerellazeae-maydis), Zonate leaf spot (Gloeocercospora sorghi).

Further plant pathogenic fungi include Plasmodiophoromycota, such asPlasmodiophora brassicae (clubroot of crucifers), Spongosporasubterranea, Polymyxa graminis, Oomycota, such as Bremia lactucae (downymildew of lettuce), Peronospora (downy mildew) in snapdragon (P.antirrhini), onion (P. destructor), spinach (P. effusa), soybean (P.manchurica), tobacco (“blue mold”; P. tabacina) alfalfa and clover (P.trifolium), Pseudoperonospora humuli (downy mildew of hops), Plasmopara(downy mildew in grapevines) (P. viticola) and sunflower (P. halstedii),Sclerophthora macrospora (downy mildew in cereals and grasses), Pythium(for example damping-off of Beta beet caused by P. debaryanum),Phytophthora infestans (late blight in potato and in tomato and thelike), Albugo spec., Ascomycota, such as Microdochium nivale (snow moldof rye and wheat), Fusarium, Fusarium graminearum, Fusarium culmorum(partial ear sterility mainly in wheat), Fusarium oxysporum (Fusariumwilt of tomato), Blumeria graminis (powdery mildew of barley (sp.hordei) and wheat (f. sp. tritici)), Erysiphe pisi (powdery mildew ofpea), Nectria galligena (Nectria canker of fruit trees), Uncinulanecator (powdery mildew of grapevine), Pseudopeziza tracheiphila (redfire disease of grapevine), Claviceps purpurea (for example, rye andgrasses), Gaeumannomyces graminis (take-all on wheat, rye and othergrasses), Magnaporthe grisea, Pyrenophora graminea (leaf stripe ofbarley), Pyrenophora teres (net blotch of barley), Pyrenophoratritici-repentis (leaf blight of wheat), Venturia inaequalis (applescab), Sclerotinia sclerotium (stalk break, stem rot), Pseudopezizamedicaginis (leaf spot of alfalfa, white and red clover),Basidiomycetes, such as Typhula incarnata (typhula blight on barley,rye, wheat), Ustilago maydis (blister smut on maize), Ustilago nuda(loose smut on barley), Ustilago tritici (loose smut on wheat, spelt),Ustilago avenae (loose smut on oats), Rhizoctonia solani (rhizoctoniaroot rot of potato), Sphacelotheca spp. (head smut of sorghum),Melampsora lini (rust of flax), Puccinia graminis (stem rust of wheat,barley, rye, oats), Puccinia recondita (leaf rust on wheat), Pucciniadispersa (brown rust on rye), Puccinia hordei (leaf rust of barley),Puccinia coronata (crown rust of oats), Puccinia striiformis (yellowrust of wheat, barley, rye and a large number of grasses), Uromycesappendiculatus (brown rust of bean), Sclerotium rolfsii (root and stemrots of many plants), Deuteromycetes (Fungi imperfecti), such asSeptoria (Stagonospora) nodorum (glume blotch) of wheat (Septoriatritici), Pseudocercosporella herpotrichoides (eyespot of wheat, barley,rye), Rynchosporium secalis (leaf spot on rye and barley), Alternariasolani (early blight of potato, tomato), Phoma betae (blackleg on Betabeet), Cercospora beticola (leaf spot on Beta beet), Alternariabrassicae (black spot on oilseed rape, cabbage and other crucifers),Verticillium dahliae (verticillium wilt), Colletotrichum, such asColletotrichum lindemuthianum (bean anthracnose), Phoma lingam (blacklegof cabbage and oilseed rape), Botrytis cinerea (grey mold of grapevine,strawberry, tomato, hops and the like).

Preferred fungal diseases to be prevented and the correspondingcausative pathogens which can be combated based on the disclosure of thepresent invention in a crop plant of interest are selected from a fungusfrom the order of Pleosporales, comprising E. turcicum/H. turcicumcausing northern corn leaf blight (NCLB), particularly affecting maizeand wheat plants, or comprising Bipolaris maydis causing southern cornleaf blight, the order of Pucciniales causing rust disease, comprisingPuccinia sorghi causing common rust, or Diploida macrospora causingDiploida leaf streak/blight, or Colletotrichum graminicola causingAnthracnose, or Fusarium spp., preferably Fusarium verticilioidescausing Fusarium stalk rot, or Gibberella spp., e.g., Gibberella zeaecausing Giberella stalk rot, or Sphacelotheca reiliana causing maizehead smut are thus plant diseases caused by pathogenic fungi which canbe prevented in the plants and by the methods of the present invention.

In one embodiment according to the various aspects of the presentinvention the at least one gene encoding at least one wall-associatedkinase may be stably integrated into the genome of the at least oneplant cell, tissue, organ, or whole plant, or the at least one geneencoding at least one wall-associated kinase may transiently introducedinto a plant cell, tissue, organ, or whole plant.

In another embodiment according to the various aspects of the presentinvention at least one further gene encoding at least one enzyme withinthe signalling cascade downstream of a wall-associated kinase may bestably integrated into the genome of the at least one plant cell,tissue, organ, or whole plant, or the at least one further gene encodingat least one enzyme within the signalling cascade downstream of awall-associated kinase may transiently introduced into a plant cell,tissue, organ, or whole plant.

Methods for introducing a gene of interest into a plant cell of interestby means of molecular biology or conventional and modern breeding andassociated tools and methodologies are disclosed herein and are known tothe skilled person.

In one embodiment, the transient introduction may comprise the directintroduction of an amino acid effector instead of the introduction of agene of interest.

In one embodiment according to the various aspects of the presentinvention the at least one gene encoding at least one wall-associatedkinase may be stably integrated into the genome of the at least oneplant cell, tissue, organ, or whole plant, wherein the introduction ofthe at least one gene encoding at least one wall-associated kinasecomprises the introgression of the at least one gene during plantbreeding.

Any of a number of standard breeding techniques can be used, dependingupon the species to be crossed. Since expression of the genes or nucleicacids of the invention may lead to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a transgenic or mutant plant of theinvention and another plant. The desired effects, e.g., expression ofthe at least one WAK gene or a mutant allele of the invention to producea plant having a modified BXD synthesis profile, or a modulated Bx1,Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9, Bx10, Bx11, Bx12, Bx13, Bx14, Igl,Glu1, Glu2, OPR2, LOX3 or AOC1 profile, can be enhanced when bothparental plants express the genes or mutant alleles of the invention, orif both allels are modified or even deleted, depending on the target tobe modified in accordance with the disclosure of the present invention.The desired effects can be passed to future plant generations bystandard propagation means. “Introgressing”, as also detailed above,thus means the integration of a gene or allele in a plant's genome bynatural means, i.e. by crossing a plant comprising the gene or allele ofinterest described herein with a plant not comprising said gene orallele. The offspring can be selected for those comprising the gene orallele of interest.

Furthermore, the methods of the present invention can result in thecreation or provision of a plant material, comprising grains or seeds,relating to any means known in the art to produce further plants, plantparts or seeds and includes inter alia vegetative reproduction methods,such as, for example, air or ground layering, division, (bud) grafting,micropropagation, stolons or runners, storage organs such as bulbs,corms, tubers and rhizomes, striking or cutting, or twin-scaling, sexualreproduction, comprising crossing with another plant, and asexualreproduction, such as e.g. apomixis, somatic hybridization and the like.

In one embodiment according to the various aspects of the presentinvention, the modification of the at least one gene encoding at leastone wall-associated kinase within step (ii) (a) or (ii) (b) of themethod for producing a plant having increased fungal resistance, or amodification of a gene encoding Bx1, Bx2, Bx3, Bx4, Bx5, Bx7, Bx8, Bx9,Bx10, Bx11, Bx12, Bx13, Bx14, Igl, Glu1, Glu2, OPR2, LOX3 or AOC1, maybe performed by at least one of a site-specific nuclease (SSN) or acatalytically active fragment thereof, or a nucleic acid sequenceencoding the same, oligonucleotide directed (ODM) mutagenesis (ODM),chemical mutagenesis, or TILLING.

TILLING, initially a functional genomics tool in model plants, has beenextended to many plant species and become of paramount importance toreverse genetics in crops species. A major recent change to TILLING hasbeen the application of next-generation sequencing (NGS) to the process,which permits multiplexing of gene targets and genomes. NGS willultimately lead to TILLING becoming an in silico procedure. Because itis readily applicable to most plants, it remains a dominantnon-transgenic method for obtaining mutations in known genes and thusrepresents a readily available method for non-transgenic approachesaccording to the methods of the present invention. As it is known to theskilled person, TILLING usually comprises the chemical mutagenesis,e.g., using ethyl methanesulfonate (EMS), or UV light inducedmodification of a genome of interest, together with a sensitive DNAscreening-technique that identifies single base mutations in a targetgene, wherein the target gene may encode a protein being selected fromthe group of a receptor-like kinase, such as a WAK, an enzyme involvedin benzoxazinoid synthesis or metabolism, defense, the lignin pathway,the jasmonic acid synthesis pathway, or a transcription factor involvedin one of the aforementioned metabolic and/or signalling pathways.

SSNs and ODM mutagenesis both are suitable techniques for precisiongenome engineering in plant cells. As it is known to the skilled person,ODM offers a rapid, precise and non-transgenic breeding alternative fortrait improvement in agriculture to address this urgent need. ODM is aprecision genome editing technology, which uses oligonucleotides to maketargeted edits in plasmid, episomal and chromosomal DNA of plantsystems.

In one embodiment according to the various aspects and embodiments ofthe present invention, the at least one site-specific nuclease (SSN), orthe nucleic acid sequence encoding the same, may be selected from atleast one of a CRISPR nuclease, including Cas or Cpf1 nucleases, aTALEN, a ZFN, a meganuclease, a base editor complex, a restrictionendonuclease, including Fold or a variant thereof, or two site-specificnicking endonucleases, or a variant or a catalytically active fragmentthereof. Said targeted genome engineering SSNs can be suitable for both,the introduction of a gene of interest not yet present in a specificgenotype, as well as the targeted mutagenesis of a gene of a givenspecific genotype to modulate (up- or downregulate) the activity of anenzyme encoded by a gene of interest to be modified in a highly preciseway.

SSNs meanwhile emerged as indispensable prerequisite for site-directedgenome engineering. SSNs are (programmable) nucleases, which can be usedto break a nucleic acid of interest at a defined position to induceeither a double-strand break (DSB) or one or more single-strand breaks.Alternatively, said nucleases can be chimeric or mutated variants, nolonger comprising a nuclease function, but rather operating asrecognition molecules in combination with another enzyme. Thosenucleases or variants thereof are thus key to any gene editing or genomeengineering approach. In recent years, many suitable nucleases,especially tailored endonucleases have been developed comprisingmeganucleases, a base editor complex, zinc finger nucleases, TALEnucleases, and CRISPR nucleases, comprising, for example, Cas, Cpf1,CasX or CasY nucleases as part of the Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR) system. The use of those SSNs and thenecessary accessory molecules, for example crRNAs, tracrRNAs, or gRNAs,and delivery systems are thus envisaged for performing the methodsaccording to the present invention.

A “base editor” as used herein refers to a protein or a fragment thereofhaving the same catalytical activity as the protein it is derived from,which protein or fragment thereof, alone or when provided as molecularcomplex, referred to as base editing or base editor complex herein, hasthe capacity to mediate a targeted base modification, i.e., theconversion of a nucleotide base of interest resulting in a pointmutation of interest which in turn can result in a targeted mutation, ifthe base conversion does not cause a silent mutation, but rather aconversion of an amino acid encoded by the codon comprising the positionto be converted with the base editor. Preferably, the base editor istemporarily or permanently linked to at least one site-specificeffector, or optionally to a component of at least one site-specificeffector complex. The linkage can be covalent and/or non-covalent.Multiple publications have shown targeted base conversion, primarilycytidine (C) to thymine (T), using a CRISPR/Cas9 nickase ornon-functional nuclease linked to a cytidine deaminase domain,Apolipoprotein B mRNA-editing catalytic polypeptide (APOBEC1), e.g.,APOBEC derived from rat. The deamination of cytosine (C) is catalyzed bycytidine deaminases and results in uracil (U), which has thebase-pairing properties of thymine (T). Most known cytidine deaminasesoperate on RNA, and the few examples that are known to accept DNArequire single-stranded (ss) DNA.

A “CRISPR nuclease” according to the present invention can be aCRISPR-based nuclease, or the nucleic acid sequence encoding the same,which is selected from the group consisting of (a) Cas9, includingSpCas9, SaCas9, SaKKH-Cas9, VQR-Cas9, St1Cas9, or (b) Cpf1, includingAsCpf1, LbCpf1, FnCpf1, (c) CasX, or (d) CasY, or any variant orderivative of the aforementioned CRISPR-based nucleases, or aCRISPR-based nuclease comprising a mutation in comparison to therespective wild-type sequence so that the resulting CRISPR-basednuclease is converted to a single-strand specific DNA nickase, or to aDNA binding effector lacking all DNA cleavage ability. A “CRISPR(-based)nuclease”, as used herein, is thus any nuclease which has beenidentified in a naturally occurring CRISPR system, which hassubsequently been isolated from its natural context, and whichpreferably has been modified or combined into a recombinant construct ofinterest to be suitable as tool for targeted genome engineering. AnyCRISPR-based nuclease can be used and optionally reprogrammed oradditionally mutated to be suitable for the various embodimentsaccording to the present invention as long as the original wild-typeCRISPR-based nuclease provides for DNA recognition, i.e., bindingproperties. Said DNA recognition can be PAM dependent. CRISPR nucleaseshaving optimized and engineered PAM recognition patterns can be used andcreated for a specific application. The expansion of the PAM recognitioncode can be suitable to target the site-specific effector complexes to atarget site of interest, independent of the original PAM specificity ofthe wild-type CRISPR-based nuclease. Cpf1 variants can comprise at leastone of a S542R, K548V, N552R, or K607R mutation, preferably mutationS542R/K607R or S542R/K548V/N552R in AsCpf1 from Acidaminococcus (cf. SEQID NO: 24). Furthermore, modified Cas variant, e.g., Cas9 variants, canbe used according to the methods of the present invention as part of abase editing complex, e.g. BE3, VQR-BE3, EQR-BE3, VRER-BE3, SaBE3,SaKKH-BE3 (see Kim et al., Nat. Biotech., 2017, doi:10.1038/nbt.3803).Therefore, according to the present invention, artificially modifiedCRISPR nucleases are envisaged, which might indeed not be any“nucleases” in the sense of double-strand cleaving enzymes, but whichare nickases or nuclease-dead variants, which still have inherent DNArecognition and thus binding ability. Other suitable Cpf1-basedeffectors for use in the methods of the present invention are derivedfrom Lachnospiraceae bacterium (LbCpf1, e.g., NCBI Reference Sequence:WP_051666128.1), or from Francisella tularensis (FnCpf1, e.g.,UniProtKB/Swiss-Prot: A0Q7Q2.1). Variants of Cpf1 are known (cf. Gao etal., BioRxiv, http://dx.doi.org/10.1101/091611). Variants of AsCpf1 withthe mutations S542R/K607R and S542R/K548V/N552R that can cleave targetsites with TYCV/CCCC and TATV PAMs, respectively, with enhancedactivities in vitro and in vivo are thus envisaged as site-specificeffectors according to the present invention. Genome-wide assessment ofoff-target activity indicated that these variants retain a high level ofDNA targeting specificity, which can be further improved by introducingmutations in non-PAM-interacting domains. Together, these variantsincrease the targeting range of AsCpf1 and thus provide a usefuladdition to the CRISPR/Cas genome engineering toolbox.

Due to the fact that receptor-like kinases and BX enzymes (cf. SEQ IDNOs: 10 to 13 and 16 to 21), Igl (SEQ ID NOs: 14 and 15), OPR2 (SEQ IDNOs:22 and 23), LOX3 (SEQ ID NOs:24 and 25), and AOC1 (SEQ ID NOs:26 and27) are ubiquitously found in a variety of plants, particularlymonocotyledonous plants (monocots) and dicotyledonous plants (dicots) ofagronomic interest, the methods according to the present invention canbe used for the targeted optimization of several important monoct anddicot crop plants.

In one embodiment according to the various aspects of the presentinvention, the at least one plant cell, tissue, organ, or whole plantprovided in step (i) (a) may be selected from the group consisting ofHordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharumofficinarium, Zea spp., including Zea mays, Setaria italica, Oryzaminuta, Oryza sativa, Oryza australiensis, Oryza alta, Triticumaestivum, Triticum durum, Secale cereale, Triticale, Malus domestica,Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucusglochidiatus, Beta spp., including Beta vulgaris, Daucus pusillus,Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotianasylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotianabenthamiana, Solanum lycopersicum, Solanum tuberosum, Coffea canephora,Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus,Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsisthaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardaminenexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsispumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassicarapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Erucavesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populustrichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicerarietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius,Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp.,Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa,Allium fistulosum, Allium sativum, Helianthus annuus, Helianthustuberosus and Allium tuberosum, or any variety or subspecies belongingto one of the aforementioned plants, preferably wherein the plant cell,tissue, organ, or whole plant in step (i) is selected from Zea mays orTriticum spp., or any variety or subspecies belonging to one of theaforementioned plants.

In one aspect of the present invention, there is thus disclosed a plantcell, tissue, organ, whole plant or plant material, or a derivative or aprogeny thereof, obtainable by any one of the methods according to thevarious aspects disclosed herein. The plant cell, tissue, organ, wholeplant or plant material, or a derivative or a progeny thereof obtainedaccording to the present invention will have at least one optimizedagronomic trait, wherein this trait is disease resistance or tolerance,preferably fungus resistance or tolerance, more preferably resistance ortolerance against NCLB caused by E. turcicum or a related fungaldiseases caused by any one of the related fungal pathogens disclosedherein. Based on the disclosure provided herein demonstrating thefunctional mechanism of a WAK induced quantitative NCLB resistance, saidresistance being associated with a reduction of the BXD biosynthesis,which in turn inhibits the hemibiotrophic fungus E. turcicum and relatedfungi, the teachings provided herein can be used to provide a plantcell, tissue, organ, whole plant or plant material, or a derivative or aprogeny thereof having a favourable BXD content, preferably a reducedBXD content, so that the plant cell, tissue, organ, whole plant or plantmaterial, or a derivative or a progeny thereof has an increasedresistance against fungal infection, i.e., fungal infestation andpersistence.

In yet a further embodiment according to the present invention, morethan one agronomic property of a plant cell or plant of interest can bemodified in addition to the introduction, modulation and/or modificationof a WAK or WAK gene of interest. Said agronomic properties are selectedfrom seed emergence, vegetative vitality, stress tolerance, diseaseresistance or tolerance against a further fungus, or against anotherpathogen, comprising a virus, bacterium, a nematode, an insect etc.,herbicide resistance, branching tendency, flowering time, seed clusters,seed density, stability and storability, threshing capability (uniformripening), lodging resistance, increased yield (seed size, yield etc.),or a modified composition of a molecule of agronomic importance (e.g.starch, carbohydrate, protein etc.) of interest, and the like.

In another aspect according to the present invention there is providedmethod for identifying at least one gene involved in increased pathogenresistance, preferably increased fungal resistance, in a plant cell,tissue, organ, whole plant, or plant material the method comprising: (i)determining the genotype of at least one plant cell, tissue, organ,whole plant, or plant material with respect to the presence of at leastone gene encoding a wall-associated kinase in the genome of said plantcell, tissue, organ, whole plant or plant material; (ii) optionally:determining the benzoxazinoid signature of the at least one plant cell,tissue, organ, whole plant, or plant material of step (i); (iii)exposing the at least one plant cell, tissue, organ, whole plant, orplant material of step (i) or (ii) to a stimulus, optionally wherein thestimulus is correlated with the benzoxazinoid signature in the at leastone plant cell, tissue, organ, whole plant, or plant material,preferably wherein the stimulus is associated with a fungal pathogeninfection; (iv) performing an analysis of at least one analyte obtainedfrom the at least one plant cell, tissue, organ, whole plant, or plantmaterial of step (i) or (ii) after exposition to the stimulus; (v)determining at least one gene being regulated upon exposition to astimulus according to step (iii) in at least one cell of the at leastone plant cell, tissue, organ, whole plant, or plant material asderivable from the analysis of at least one analyte as defined in step(iv), (vi) subjecting the at least one gene as determined in step (v) toa functional characterization; and (vii) providing at least one geneinvolved in increased pathogen resistance, preferably increased fungalresistance, in a plant cell, tissue, organ, whole plant, or plantmaterial.

In one embodiment, the determination of the genotype of at least oneplant cell, tissue, organ, whole plant, or plant material with respectto the presence of at least one gene encoding a wall-associated kinasemay be performed by determining in the genome of a plant cell, tissue,organ, whole plant, or plant material of interest the presence and/ortranscript level of a WAK gene of interest, preferably a WAK genecomprising a nucleotide sequence according to SEQ ID NO: 1 or 7, orcomprising a nucleotide sequence having at least 60%, 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to one of thenucleotide sequence according to SEQ ID NO: 1 or 7, preferably over theentire length of the sequence, or comprising a nucleotide sequencehybridizing with a nucleotide sequence complementary to the nucleotidesequence according to SEQ ID NO: 1 or 7 preferably under stringentconditions, or comprising a nucleotide sequence encoding for an aminoacid sequence of SED ID NO: 2 or 8 or for an amino acid sequence havingat least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to one of SEQ ID NO: 2 or 8.

In another embodiment, determining the benzoxazinoid signature comprisesa step of determination of the presence and/or the transcript level ofat least one gene from the BXD biosynthesis pathway and/or the jasmonicacid pathway, The gene may be selected from any one of SEQ ID NOs: 10,12, 14, 16, 18, 20, 22, 24 or 26, or a variant, homologous gene, allelor mutant or a fragment thereof. Bioinformatic tools for thedetermination and/or alignment of sequences of interest are disclosedherein, or are readily available to the skilled person.

In one embodiment, the determination of the genotype of at least oneplant cell, tissue, organ, whole plant, or plant material with respectto the presence of at least one gene encoding a wall-associated kinasemay also comprise the sequencing of a gene having a certain sequenceidentity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to oneof the gene sequences disclosed herein, which gene has not yet beenannotated in a publicly available genome database to determine theprecise sequence of said gene by means of molecular biology, e.g., PCRtechniques.

In one embodiment, the method for identifying at least one gene involvedin increased pathogen resistance, preferably increased fungalresistance, in a plant cell, tissue, organ, whole plant, or plantmaterial may comprise the determination of the benzoxazinoid (BXD)signature of the at least one plant cell, tissue, organ, whole plant, orplant material of step (i). The benzoxazinoid signature means thequalitative and/or quantitative determination of at least one BXDsecondary metabolite of interest as disclosed herein. This determinationcan provide a reference value for any subsequent analysis. Thebenzoxazinoid signature determination, which can be performed atdifferent timepoints and with or without the addition of a stimulus,thus can provide information on the background level of a specific BXDpresent before and after addition of a stimulus. Furthermore, the BXDsignature may provide data on the total amount of mixed BXD compoundssynthesized in a plant, plant cell, tissue, organ or whole plant undersuitable and defined conditions. The BXD signature may thus serve asreference value to have a benchmark for any subsequent modificationsand/or modulations performed in accordance with the methods of thepresent invention. Due to the fact that BXD synthesis depends on theaction of different enzymes in the terminal branch of the synthesispathway, more than one different BXD compound may be analyzed to providea BXD signature of a plant cell, tissue, organ, or whole plant ofinterest representing a full picture of the different BXD compoundssynthesized by the plant under defined conditions (timepoint, stimulus,stimulus amount and environmental factors, for example, biotic orabiotic stress).

In one embodiment, the method for identifying at least one gene involvedin increased pathogen resistance, preferably increased fungalresistance, in a plant cell, tissue, organ, whole plant, or plantmaterial may comprise the exposition of the at least one plant cell,tissue, organ, whole plant, or plant material of step (i) or (ii) to astimulus, optionally wherein the stimulus is correlated with thebenzoxazinoid signature in the at least one plant cell, tissue, organ,whole plant, or plant material, preferably wherein the stimulus isassociated with a fungal pathogen infection.

A “stimulus” in this context refers to any naturally occurring,endogenous or exogenous, or non-naturally occurring substance chemicalsubstance stimulating a plant cell, tissue, organ, whole plant.Preferably, the stimulus is a stimulus derived from or associated with apathogen, preferably a fungal pathogen. The stimulus may be a known PAMPor DAMP triggering an immune response mediated by a receptor-like kinasein a plant cell, tissue, organ, whole plant. The correlation may be ofdirect or indirect nature. The “stimulus” may also be an endogenoussubstance, e.g., a BXD or jasmonic acid, or a synthetic variant thereof,as BXD compounds and jasmonic acids may induce feedback regulationmechanisms in a plant cell. The “stimulus” may the pathogen by itselfcausing the desired response in a plant.

The stimulus may thus be any environmental stimulus which will cause aresponse in a plant, wherein the response is effected by a signalcascade, or reaction within a plant cell, tissue, organ, whole plant,e.g., resulting in a different transcriptome profile in comparison tothe transcriptome profile of a non-stimulated plant. Preferably, thestimulus is correlated with a benzoxazinoid signature in at least oneplant cell, tissue, organ, whole plant, or plant material. In oneembodiment, where the correlation between a stimulus and the BXDsignature is not known, a correlation between a stimulus of interest andthe BXD signature can be easily determined by measuring the up- ordown-regulation of genes within the BXD signalling pathway upon additionof a stimulus of interest to determine a direct or indirect correlation.

In a preferred embodiment, the stimulus is associated with a fungalpathogen, but is not restricted thereto. As it is known in the field ofplant pathophysiology, plants evolved sophisticated strategies torespond to a stimulus as provided by a variety of different plantpathogens to initiate defense responses. Certain response may be highlyspecific for a pathogen, or one specific molecule associated or producedby said pathogen, whereas other defense strategies are part of a globalregulatory network as associated by a stimulus of interest. According tothe methods of the present invention it is thus possible to analyze theeffect of a stimulus of interest a pathway of interest to identify anyimplication in the BXD or jasmonic acid biosynthesis pathway having afavourable effect on plant fungal response as disclosed herein in ahighly targeted way to identify new target genes contributing to afavourable fungal defense response in a plant cell, tissue, organ, orwhole plant of interest.

In one embodiment, the method for identifying at least one gene involvedin increased pathogen resistance, preferably increased fungalresistance, in a plant cell, tissue, organ, whole plant, or plantmaterial according to the present invention can comprise an additionalstep of electronically transmitting and/or electronically storing dataon a computer readable medium.

An “analyte” obtained from the at least one plant cell, tissue, organ,or whole plant may comprises a nucleic acid, including DNA and RNA, anamino acid sequence, or a plant metabolite.

In one embodiment, a transcriptome analysis, i.e., an analysis of thesum total of all the messenger RNA molecules expressed from the genes ofan organism, using RNA obtained from the at least one plant cell,tissue, organ, or whole plant of step (ii) after exposition to thestimulus is performed to obtain data on any changes in the transcriptionprofile of certain genes in a plant cell, tissue, organ, whole planttreated with a stimulus of interest in comparison to plant cell, tissue,organ, or whole plant not treated with the respective stimulus. Avariety of different tools to perform a transcriptome analysis ofgenome-wide differentially expressed RNA and to analyze altered geneexpression/transcription is available to the skilled person. In oneembodiment, the determination of at least one gene being regulated uponexposition to a stimulus according to step (iii) of the above method foridentifying at least one gene involved in increased pathogen resistancein at least one cell of the at least one plant cell, tissue, organ,whole plant thus comprises the determination of the transcription levelof a gene. Preferably, differentially regulated, or highly regulatedgenes, e.g., genes being significantly up- or down-regulated incomparison to a non-treated plant or plant cell, may be furtheranalyzed.

In another embodiment, a proteome analysis, i.e., an analysis of theentire complement of proteins that is or can be expressed by a plantcell, tissue, or organism, using amino acids obtained from the at leastone plant cell, tissue, organ, or whole plant of step (ii) afterexposition to the stimulus is performed to obtain data on any changes inthe transcription profile in a plant cell, tissue, organ, whole planttreated with a stimulus of interest in comparison to plant cell, tissue,organ, or whole plant not treated with the respective stimulus. Severalmethods for quantitative and qualitative proteome analysis, of the wholeproteome or parts thereof, are available to the skilled person.

In yet another embodiment, an analysis of a metabolite, e.g., asubstance produced by the at least one plant cell, tissue, organ orwhole plant and representing an intermediate or product of itsmetabolism, is performed to identify the effect of a stimulus has on theoverall constitution and production level with respect to saidmetabolite of interest.

In one embodiment of the method for identifying at least one geneinvolved in increased pathogen resistance, preferably increased fungalresistance, in a plant cell, tissue, organ, whole plant, or plantmaterial, a gene of interest determined, said gene being regulated uponexposition to a stimulus, preferably a stimulus influencing a BXDsignature, may be subjected to a functional characterization. Thefunctional characterization may comprise an in silico analysis, an invitro analysis, an in vivo analysis, or a combination of theaforementioned analyses. The in silico analysis may comprise thedetermination of any known function of said gene in different plant, orinformation on available allelic variants of said gene in differentplants or different germplasm. Furthermore, the in silico analysis maycomprise the determination of the locus of a gene such determined in thegenome of a plant of interest, or the determination of regulatorysequences associated with the gene of interest. An in vitro analysis ormanipulation may comprise the cloning, sequencing and characterizationof the gene of interest and/or the creation of an expression construct,or vector, or a fusion construct, or the creation of mutants of a geneof interest. An in vitro analysis or manipulation may further comprisethe introduction of a gene of interest, comprised by a suitableconstruct, into a target plant, tissue, organ or whole plant of interestby a suitable delivery vector. The in vivo analysis may comprise theanalysis of different plants or plant cells, tissues or organs fromdifferent species, cultivars or varieties comprising or not comprisingthe gene of interest in their genome to provide a functionalcharacterization of the phenotype the gene of interest may participatein, optionally by subjecting the different plants or plant cells,tissues or organs from different species, cultivars or varieties todifferent stimuli and controlled conditions to be able to compare therespective results.

In one embodiment, at least one gene involved in increased pathogenresistance as identified according to the methods of the presentinvention can be further subjected to directed mutagenesis studies andsubsequent functional analyses to identify mutations positively ornegatively effecting a phenotype of interest, wherein the phenotype is achange in the BXD signature, or a change of fungal resistance incomparison to the respective wild-type. Methods to introduce (multiple)site-directed mutations into a given gene of interest are available tothe skilled person.

In one aspect of the present invention there is provided a plant cell,tissue, organ, whole plant or plant material, or a derivative or aprogeny thereof, obtainable by introducing at least one gene as providedby the method for identifying at least one gene involved in increasedpathogen resistance, preferably increased fungal resistance into thegenome of at least one cell of at least one of a plant cell, tissue,organ, or whole plant.

In one embodiment, the plant cell, tissue, organ, whole plant or plantmaterial, or a derivative or a progeny thereof, comprises at least onewall-associated kinase (WAK) selected from Htn1, Ht2, or Ht3, or anallelic variant, a mutant or a functional fragment thereof, or a geneencoding the same, preferably wherein the at least one wall-associatedkinase a) is encoded by a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 1 or 7, or a functional fragmentthereof, b) is encoded by a nucleic acid molecule comprising thenucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to nucleotide sequence of SEQ ID NO: 1or 7, preferably over the entire length of the sequence, c) is encoded anucleic acid molecule hybridizing with a complementary sequence to a) orb) under stringent conditions, d) is encoded by a nucleic acid moleculecomprising the nucleotide sequence coding for an amino acid sequence ofSEQ ID NO: 2 or 8, or a functional fragment thereof, e) is encoded by anucleic acid molecule comprising the nucleotide sequence coding for anamino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% sequence identity to amino acid sequence of SEQ ID NO: 2or 8, preferably over the entire length of the sequence, f) comprisingthe amino acid sequence of SEQ ID NO: 2 or 8, or g) comprising an aminoacid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% sequence identity to amino acid sequence of SEQ ID NO: 2 or8, preferably over the entire length of the sequence, provided that thesequence, optionally after expression, still encodes at least onefunctional Htn1, Ht2, or Ht3, or an allelic variant, a mutant, or afunctional fragment thereof. In a preferred embodiment, the plant cell,tissue, organ, whole plant or plant material, or a derivative or aprogeny thereof, comprises at least one wall-associated kinase or anallelic variant, a mutant or a functional fragment thereof, or a geneencoding the same, which has been introduced or introgressed, or whichat least one wall-associated kinase or an allelic variant, a mutant or afunctional fragment thereof, or a gene encoding the same, comprises atleast one mutation enhancing the kinase activity of the at least oneWAK.

In another preferred embodiment, the plant cell, tissue, organ, wholeplant or plant material, or a derivative or a progeny thereof, comprisesat least one further introduced or introgressed enzyme, or the geneencoding the same, wherein the at least one further gene or enzyme isselected from a bx1, bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 gene(SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24 or 26, respectively), or ahomologous gene thereof, or the respective proteins encoded by saidgenes (SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25 or 27, respectively),or a homolog thereof or an allelic variant or mutant thereof, preferablya mutant resulting in decreased transcription and/or translation of thebx1, bx2, igl, bx6, bx11, bx14, opr2, lox3 or aoc1 gene or protein,respectively. The at least one mutation may thus reside in a regulatoryregion of such a gene leading to a reduced transcription, or themutation may result in at least one point mutation affecting thecatalytic activity of the translated protein so that said protein orenzyme has a decreased capability to synthesize a BXD compound.

In one embodiment, the introduction of at least one gene into plantcell, tissue, organ, whole plant or plant material, obtainable byintroducing at least one gene as provided by the method for identifyingat least one gene involved in increased pathogen resistance, preferablyincreased fungal resistance, is a stable introduction, preferably astable introduction mediated by plant breeding, or a stable introductionmediated by means of molecular biology, comprisingAgrobacterium-mediated transformation, genome editing, or a combinationthereof.

In one embodiment, the introduction may be effected by introgression ofthe at least one gene identified, and/or the introduction may beeffected may any means of molecular biology. In one embodiment theintroduction of a gene or allele determined can take place byrecombination between two donor genomes, e.g., in a fused protoplast,wherein at least the donor protoplast carries the gene allele ofinterest in its genome. In any case, any progeny or derivativescomprising the gene allele of interest can then be subjected to repeatedback-crossing steps with a plant line carrying a genetic background ofinterest to select for the gene allele of interest in the resultingderivatives or progeny. The result may be the fixation of the geneallele of interest such introgressed in a selected genetic background.The whole process of introgression can, for example, take place by amixture of breeding strategies and techniques of molecular biology toachieve at a genotype/phenotype of interest for a given germplasm,plant, plant cell or plant material.

In one embodiment, there is thus provided an improved donor source ofgermplasm having, e.g. by introgression, enhanced resistance to a fungusof interest, preferably wherein the fungus resistance against whichresistance is increased, or the disease caused by said fungus isselected from a fungus of the order of Pleosporales, comprising E.turcicum/H. turcicum causing northern corn leaf blight (NCLB),particularly affecting maize and wheat plants, southern corn leaf blight(Bipolaris maydis), the order of Pucciniales causing rust disease,comprising common rust (Puccinia sorghi), or Diploida leaf streak/blight(Diploida macrospora/Stenocarpella macrospora), or Colletotrichumgraminicola, or Fusarium spp., preferably Fusarium verticilioidescausing Fusarium stalk rot, or Gibberella spp., e.g., Gibberella zeaecausing Giberella stalk rot, rust, stalk rot, maize head smut(Sphacelotheca reiliana), and Diploida leaf streak/blight. Thisgermplasm can then serve as basis for further breeding steps.

In another embodiment, the introduction of at least one gene asidentified and provided by the method for identifying at least one geneinvolved in increased pathogen resistance into at least one plant cell,tissue, organ, whole plant may be effected by at least one means ofmolecular biology, comprising the use of a delivery vehicle or vector.Optionally, the method can further comprise the modification ormodulation of a gene of interest using at least one of a site-specificnuclease (SSN) or a catalytically active fragment thereof, or a nucleicacid sequence encoding the same, oligonucleotide directed mutagenesis,chemical mutagenesis, or TILLING, wherein the at least one site-specificnuclease (SSN), or the nucleic acid sequence encoding the same, isselected from at least one of a CRISPR nuclease, including Cas or Cpf1nucleases, a TALEN, a ZFN, a meganuclease, a base editor complex, arestriction endonuclease, including Fok1 or a variant thereof, or twosite-specific nicking endonucleases, or a variant or a catalyticallyactive fragment thereof.

In yet a further aspect according to the present invention, there isprovided a method of increasing pathogen resistance, preferably fungalresistance, in a plant cell, tissue, organ, whole plant, or plantmaterial, the method comprising: (i) providing at least one plant cell,tissue, organ, whole plant or plant material; (ii) (a) treating the atleast one plant cell, tissue, organ, whole plant or plant materialaccording to step (i) with a substance neutralizing the effect of atleast one benzoxazinoid, and/or (ii) (b) treating the at least one plantcell, tissue, organ, whole plant or plant material according to step (i)with a substance activating the signalling pathway downstream of atleast one wall-associated kinase; and/or (ii) (c) treating the at leastone plant cell, tissue, organ, whole plant or plant material accordingto step (i) with a substance modulating or modifying the activity of atleast promoter or at least one regulatory sequence of at least one geneof the at least one plant cell, tissue, organ, whole plant or plantmaterial of step (i), wherein said at least promoter or at least oneregulatory sequence is involved in the regulation of transcription of atleast on gene involved in the signalling pathway of, or downstream of atleast one wall-associated kinase or involved in the synthesis pathway ofat least one benzoxazinoid; (ii) (d) treating the at least one plantcell, tissue, organ, whole plant or plant material according to step (i)with a substance inhibiting the synthesis of at least one benzoxazinoid;(iii) reducing the amount of at least one benzoxazinoid and therebyincreasing pathogen resistance, preferably fungal resistance, in atleast one plant cell, tissue, organ, whole plant, or plant material.

A “a substance neutralizing the effect of at least one benzoxazinoid” asused herein is to be construed broadly and comprises any naturallyoccurring or synthetic molecule, which can interact with a BXD compoundto decrease the natural effect of the BXD compound said BXD compoundwould exert (endogenously and/or exogenously) on a plant or the plantenvironment. Preferably, the substance neutralizing the effect of atleast one benzoxazinoid can be added to a plant cell, tissue, organ, orwhole plant, optionally coated or together with a suitable deliveryvehicle, so that the substance can be transferred into a plant cell ofinterest. Alternatively, the substance can be added to a plant cell,tissue, organ or whole plant to neutralize the effect of a volatilecompound released by a plant cell, tissue, organ or whole plant.Preferably, the substance neutralizing the effect of at least onebenzoxazinoid is not toxic to the plant cell, tissue, organ, or wholeplant, or to the environment. As it is known that jasmonic acid (JA)treatment can induce the accumulation of BXD compounds (Oikawa et al,2002 and 2004), a substance according to the present invention may alsobe a substance scavenging or reducing the amount of jasmonic acid todecrease the accumulation of a BXD compound, which in turn leads to theincreased fungal resistance of a plant cell, tissue, organ or wholeplant of interest. The substance may also interfere with thetranscription of at least one Bx, Igl or a further gene involved in theBXD or jasmonic acid biosynthesis pathway.

In a further embodiment, alone or in combination with the use of aneutralizing substance, at least one plant cell, tissue, organ, wholeplant or plant material can be treated with a substance activating thesignalling pathway downstream of at least one wall-associated kinase. Asused in the context of molecular biology, the terms “upstream” and“downstream” can refer to the temporal and mechanistic order of cellularand molecular events. For example, in signal transduction cascade, thesecond messenger or an intracellular kinase acts downstream to—that isto say, temporally after—activation of cell membrane receptors, forexample a WAK. The other way around, activation of cell membranereceptors occurs upstream of—that is to say, prior to—the production ofsecond messengers or the activation or inhibition of further enzymesacting later and intracellularly in the signalling cascade. Such anactivating substance can be selected from a substance acting from theexterior of a plant or plant cell, for example, a substance being a PAMPor DAMP for a receptor-like kinase, e.g., a WAK, so that a strongersignal is received and the receptor mediated response is enhanced.Furthermore, the substance may activate the kinase function of a WAK, orany kinase downstream of the WAK. Finally, the substance may act at theinterface between the WAK and a further BXD or jasmonic acidbiosynthesis pathway.

For the wheat WAK gene TaWAK/Snn1 it was shown that it is hijacked bythe necrotrophic effector SnTox1 that triggers programmed cell deathallowing a pathogen to feed and grown on the dead tissue (Shi et al.2016). Furthermore, these data show that elicitors recognized by WAKscan both be cell wall derived degraded polysaccharides (e.g. OGs) orpathogenic short peptides (SnTox1) (Brutus et al. 2010; Shi et al.2016). Thus, there is increasing evidence for a complex nature andfunctional divergence of WAKs in perception of types of ligands and intheir role of interacting with biotic diseases in a direct as well as anindirect way. Preferably, an activating substance according to thepresent invention is a substance directly activating a WAK of interestwhich in turn, directly or indirectly, leads to a decreased synthesis ofat least one BXD compound, which in turn increases the fungal resistanceof a plant cell, tissue, organ or whole plant.

The inventors of the present invention demonstrated that the WAKZmWAK-RLK1 functions upstream of the BXDs biosynthesis pathway anddecreases the content of secondary metabolites BXDs compounds, e.g.,DIM₂BOA-Glc. As the BXD class of secondary metabolites has been found inmany of cereal species such maize, wheat and rice, which are the mostimportant food crops worldwide, the methods according to the variousaspects of the present invention can thus be used to effect the WAKsignaling cascade intrinsically linked to the BXD synthesis, which inturn was found to be key to provide new strategies in fungal defense inplants, preferably for reducing susceptibility to northern corn leafblight already at the seeding stage. For example, the storage glucosideDIM₂BOA-Glc was found to be constantly lower in susceptible ZmWAK-RLK1mutants, which suggested DIM₂BOA-Glc severed as a candidatesusceptibility compound for promoting E. turcicum infection. Knock-outof this compound has been shown to slightly increase the performance ofcorn leaf aphids Rhopalosiphum maidis (Handrick, Vinzenz, et al.“Biosynthesis of 8-O-methylated benzoxazinoid defense compounds inmaize.” The Plant Cell 28.7 (2016): 1682-1700), a completely differentclass of plant pathogens, not infecting, yet feeding on a plant, whereasthe functional mechanism of DIM₂BOA-Glc in interaction withphloem-feeding insects as presently known is possibly different andantagonistic. The methods and findings according to the presentinvention and mainly the new insights in gap bridge of WAKs and thesecondary defense metabolite BXDs can also be used to provide newdefense mechanisms against aphids and other phloem feeding insects to aplant, preferably a crop plant.

In one preferred embodiment, the methods comprise the modulation ormodification of at least one further gene from a BXD and/or jasmonicacid biosynthesis pathway as disclosed herein to further decrease thecontent of secondary metabolites BXDs compounds, e.g., DIM₂BOA-Glc andthus to enhance fungal resistance in a plant.

In yet a further embodiment of the method of increasing pathogenresistance, preferably fungal resistance, in a plant cell, tissue,organ, whole plant, or plant material according to the presentinvention, the method comprises treating the at least one plant cell,tissue, organ, whole plant or plant material according to step (i) witha substance modulating the activity of at least promoter or at least oneregulatory sequence of at least one gene of the at least one plant cell,tissue, organ, whole plant or plant material of step (i), wherein saidat least promoter or at least one regulatory sequence is involved in theregulation of transcription of at least on gene involved in thesignalling pathway of, or downstream of at least one wall-associatedkinase, or involved in the synthesis pathway of at least onebenzoxazinoid. By modulating or modifying the activity of a promoter orregulatory sequence, the transcription level of a gene of interest andin turn the expression level of a protein of interest can be influencedin a targeted way on a molecular level, or by introducing atranscription factor, preferably a synthetic transcription factor likeTAL efector activator/repressor or CRISPR-dCas9 activator/repressor, fora given promoter/gene into a cell. In embodiments, where a promoter ismodified in a targeted way, the modification is performed by at leastone of a site-specific nuclease (SSN) or a catalytically active fragmentthereof, or a nucleic acid sequence encoding the same, oligonucleotidedirected mutagenesis, chemical mutagenesis, or TILLING.

In one embodiment, the at least one site-specific nuclease (SSN), or thenucleic acid sequence encoding the same, is selected from at least oneof a CRISPR nuclease, including Cas or Cpf1 nucleases, a TALEN, a ZFN, ameganuclease, a base editor complex, a restriction endonuclease,including Fold or a variant thereof, a recombinase, or two site-specificnicking endonucleases, or a variant or a catalytically active fragmentthereof. Preferably, a targeted point mutation is introduced modifyingthe promoter region, wherein the modification can make use of atransient introduction of the site-specific nuclease tools to obtain anon-transgenic plant cell, tissue, organ or whole plant.

In yet another or further embodiment of the method of increasingpathogen resistance, preferably fungal resistance, in a plant cell,tissue, organ, whole plant, or plant material according to the presentinvention, the method comprises treating the at least one plant cell,tissue, organ, whole plant or plant material according to step (i) witha substance inhibiting the synthesis of at least one benzoxazinoid. Asubstance inhibiting the synthesis of at least one benzoxazinoid can bea double stranded RNA (dsRNA) which is suitable to reduce the expressionlevel of at least on gene involved in the signalling pathway of, ordownstream of at least one wall-associated kinase, or involved in thesynthesis pathway of at least one benzoxazinoid, wherein by saidreduction of the expression level the synthesis or the amount of atleast one benzoxazinoid and thereby increasing pathogen resistance,preferably fungal resistance, in at least one plant cell, tissue, organ,whole plant, or plant material. This down regulating of gene expressionis well-known to a person skilled in art as RNAi approach or miRNAinterference approach (Fire, A, Xu, S, Montgomery, M, Kostas, S, Driver,S, Mello, C. (1998). Potent and specific genetic interference bydouble-stranded RNA in Caenorhabditis elegans, Nature 391 (6669):806-811). Preferably the substance inhibiting the synthesis of at leastone benzoxazinoid is at least one siRNA or an siRNA library directed toat least on gene involved in the signalling pathway of, or downstream ofat least one wall-associated kinase, or involved in the synthesispathway of at least one benzoxazinoid. The siRNA or siRNA library can bepart of one or more expression cassettes. The siRNA may comprise a firststrand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′end, wherein the first strand is complementary to at least 15nucleotides of the at least on gene involved in the signalling pathwayof, or downstream of at least one wall-associated kinase, or involved inthe synthesis pathway of at least one benzoxazinoid, and an secondstrand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′end, and wherein at least 12 nucleotides of the first and second strandsare complementary to each other and form a small interfering RNA (siRNA)duplex under physiological conditions, and wherein the siRNA silencesthe at least on gene involved in the signalling pathway of, ordownstream of at least one wall-associated kinase, or involved in thesynthesis pathway of at least one benzoxazinoid.

The various embodiments of the aspect of the present invention beingdirected to a method of increasing pathogen resistance, preferablyfungal resistance, in a plant cell, tissue, organ, whole plant, or plantmaterial, alone or in combination, may result in the targeted reductionof the amount of at least one benzoxazinoid and thereby may lead to anincreased pathogen resistance, preferably fungal resistance, in at leastone plant cell, tissue, organ, whole plant, or plant material. In yet afurther aspect according to the present invention there is thus provideduse of a substance a for increasing pathogen resistance, preferablyfungal resistance, in at least one plant cell, tissue, organ, wholeplant, or plant material. The substance may act as a plant protectiveagent and may be applied to a plant exogenously, or the substance may bea scavenger of any plant molecule or material, or the substance may actas a modulator of WAK, of the downstream signalling cascade, or of acellular pathway disclosed herein being related to the WAK signallingpathway, preferably a BXD and/or jasmonic acid biosynthesis pathway, orthe substance may act on the transcription of any gene involved in theWAK or an associated pathway as disclosed herein, wherein the substancecan thus directly or indirectly influence, preferably decrease, theamount of a BXD compound produced and stored in a plant cell. Therebythe use of the substance according to the present invention will lead toa decreased BXD level and thus an increased fungal resistance in a plantcell, tissue, organ, or whole plant of interest.

Delivery Methods:

A variety of suitable delivery techniques suitable according to themethods of the present invention for introducing genetic material into aplant cell are known to the skilled person., e.g. by choosing directdelivery techniques ranging from polyethylene glycol (PEG) treatment ofprotoplasts (Potrykus, Ingo, et al. “Direct gene transfer to cells of agraminaceous monocot.” Molecular and General Genetics MGG 199.2 (1985):183-188), procedures like electroporation (D'Halluin, Kathleen, et al.“Transgenic maize plants by tissue electroporation.” The plant cell 4.12(1992): 1495-1505), microinjection (Neuhaus, G., et al. “Transgenicrapeseed plants obtained by the microinjection of DNA intomicrospore-derived embryoids.” Theoretical and Applied Genetics 75.1(1987): 30-36), silicon carbide fiber whisker technology (Kaeppler, H.F., et al. “Silicon carbide fiber-mediated stable transformation ofplant cells.” Tag Theoretical and Applied Genetics 84.5 (1992):560-566), viral vector mediated approaches (Gelvin, Nature Biotechnology23, “Viral-mediated plant transformation gets a boost”, 684-685 (2005))and particle bombardment (see e.g. Sood et al., 2011, BiologiaPlantarum, 55, 1-15).

Despite transformation methods based on biological approaches, likeAgrobacterium transformation or viral vector mediated planttransformation, and methods based on physical delivery methods, likeparticle bombardment or microinjection, have evolved as prominenttechniques for introducing genetic material into a plant cell or tissueof interest. Helenius et al. (“Gene delivery into intact plants usingthe Helios™ Gene Gun”, Plant Molecular Biology Reporter, 2000, 18(3):287-288) discloses a particle bombardment as physical method forintroducing material into a plant cell. Currently, there thus exists avariety of plant transformation methods to introduce genetic material inthe form of a genetic construct into a plant cell of interest,comprising biological and physical means known to the skilled person onthe field of plant biotechnology and which can be applied to introduceat least one gene encoding at least one wall-associated kinase into atleast one cell of at least one of a plant cell, tissue, organ, or wholeplant. Notably, said delivery methods for transformation andtransfection can be applied to introduce the tools of the presentinvention simultaneously. A common biological means is transformationwith Agrobacterium spp. which has been used for decades for a variety ofdifferent plant materials. Viral vector mediated plant transformationrepresents a further strategy for introducing genetic material into acell of interest. Physical means finding application in plant biologyare particle bombardment, also named biolistic transfection ormicroparticle-mediated gene transfer, which refers to a physicaldelivery method for transferring a coated microparticle or nanoparticlecomprising a nucleic acid or a genetic construct of interest into atarget cell or tissue. Physical introduction means are suitable tointroduce nucleic acids, i.e., RNA and/or DNA, and proteins. Likewise,specific transformation or transfection methods exist for specificallyintroducing a nucleic acid or an amino acid construct of interest into aplant cell, including electroporation, microinjection, nanoparticles,and cell-penetrating peptides (CPPs). Furthermore, chemical-basedtransfection methods exist to introduce genetic constructs and/ornucleic acids and/or proteins, comprising inter alia transfection withcalcium phosphate, transfection using liposomes, e.g., cationicliposomes, or transfection with cationic polymers, includingDEAD-dextran or polyethylenimine, or combinations thereof. Said deliverymethods and delivery vehicles or cargos thus inherently differ fromdelivery tools as used for other eukaryotic cells, including animal andmammalian cells and every delivery method has to be specificallyfine-tuned and optimized so that a construct of interest for introducingand/or modifying at least one gene encoding at least one wall-associatedkinase in the at least one plant cell, tissue, organ, or whole plant;and/or can be introduced into a specific compartment of a target cell ofinterest in a fully functional and active way. The above deliverytechniques, alone or in combination, can be used for in vivo (in planta)or in vitro approaches.

In one embodiment, a regulatory sequence according to the presentinvention may be a promoter sequence, wherein the editing or mutation ormodulation of the promoter comprises replacing the promoter, or promoterfragment with a different promoter (also referred to as replacementpromoter) or promoter fragment (also referred to as replacement promoterfragment), wherein the promoter replacement results in any one of thefollowing or any one combination of the following: an increased promoteractivity, an increased promoter tissue specificity, a decreased promoteractivity, a decreased promoter tissue specificity, a new promoteractivity, an inducible promoter activity, an extended window of geneexpression, a modification of the timing or developmental progress ofgene expression in the same cell layer or other cell layer, for example,extending the timing of gene expression in the tapetum of anthers, amutation of DNA binding elements and/or a deletion or addition of DNAbinding elements. The promoter (or promoter fragment) to be modified canbe a promoter (or promoter fragment) that is endogenous, artificial,pre-existing, or transgenic to the cell that is being edited. Thereplacement promoter or fragment thereof can be a promoter or fragmentthereof that is endogenous, artificial, pre-existing, or transgenic tothe cell that is being edited.

The present invention will now be illustrated by the following Examples,which are not construed to limit the scope of the present invention.

EXAMPLES Example 1: Plant Material and Growth Conditions

Seventeen maize inbred lines were used, including: (1) historicalcultivars B37 and w22, and the NILs B37Htn1 and w22Htn1 that contain theNCLB resistance gene Htn1; (2) Breeding line RP3 and its NIL lineRP3Htn1 carrying Htn1 from KWS (see US 2016/0201080 A1); (3) three pairsof mutants RLK1b (S, compromising Htn1 resistance), RLK1d and RLK1f, andcorresponding sister lines RLK1b-wt (R, carrying functional Htn1),RLK1d-wt, and RLK1f-wt, which were produced by EMS-mutagenesis inRP3Htn1 (Hurni et al. 2015); (4) three maize mutants (bx1, bx2 and bx6)and parental line w22, which were provided by Prof. Georg Jander(Cornell University, Ithaca, US). Two or three maize seeds were sown ineach Jiffy pot (ø8 cm), and fifteen pots were placed in one tray.Seedling plants were grown in a greenhouse condition of 16 h at 20° C.in the day, 8 h at 18° C. in the night and approximately 60% relativehumidity.

Example 2: NCLB Infection Tests in the Greenhouse

Testing for NCLB resistance using E. turcicum isolate Passau-1 wasperformed as previously described with minor modification (Hurni et al.2015). After the second leaves had fully emerged, the newly emergedleaves were cut and removed until the end of each test experiment.Single spore inoculation and culture on PDA medium plate, harvest andquantification of progeny spores were described (Hurni et al. 2015).Instead of infection by dropping 80 μl spore suspension into the leafsheath of the second leaf twice, here maize seedlings were infected onceby spray (sprayer: ø28 mm, Semadeni, Ostermundigen, Switzerland). Each 4trays (ca. 60-80 seedlings) were sprayed with 4 ml of spore suspension(4.5×10⁴ spores/ml). A very high humidity mic-condition was produced byplacing plastic hoods on top of each tray after infection. Each plantwas scored for disease symptom between 11 and 25 days and the severitywas evaluated by calculating the area under the disease progress curve(AUDPC) or by quantifying the diseased leaf area of the inoculatedsecond leaves (PrimDLA). About 15 seedling plants were scored in eachgenotype in each experiment.

Example 3: Vector Construction and Subcellular Localization

The coding sequence of ZmWAK-RLK1 was amplified using a cDNA clone astemplate, which was initially amplified in NCLB resistance genotypeRP1Htn1 (Hurni et al. 2015). The PCR fragment was introduced into theGateway donor vector pDONR207 using the Gateway® BP Clonase® II Enzymemix (Thermo Fisher Scientific, Wilmington, USA). The generated entryvector carrying the target ZmWAK-RLK1 sequence was inserted byrecombination with the destination vector pUBC-GFP-DEST, to produce anin-frame ZmWAK-RLK1+c′-eGFP fusion protein construct driven byArabidopsis ubiqutin-10 (UBQ10) gene promotor (Grefen, Christopher, etal. “A ubiquitin-10 promoter-based vector set for fluorescent proteintagging facilitates temporal stability and native protein distributionin transient and stable expression studies.” The Plant Journal 64.2(2010): 355-365). The UBQ10::ZmWAK-RLK1-c′-eGFP construct (SEQ ID NO:9)together with a reference plasmid PIP2A-mCherry (Cutler et al., RandomGFP::cDNA fusions enable visualization of subcellular structures incells of Arabidopsis at a high frequency. Proc. Natl Acad. Sci. USA 97,3718-3723, (2000) contains 35S::PIP2A_c′_RFP construct, which islocalized to the plasma membrane) were mixed with nanograde goldparticles and co-bombarded into onion epidermal cells, which weresubsequently incubated at 20° C. in the dark for 2-3 days until beingready for observation using a confocal microscope. Plasmolysis wasinduced by adding a 0.8 M mannitol solution. Furthermore, both plasmidswere transformed into Agrobacterium GV3101 and co-infiltrated into4-week-old N. bentaniana leaves, which were ready for observation 2 dayspost infiltration. The primers used for vector construct are provided inthe below Table 1.

TABLE 1 Forward (F) and reverse (R) primers used for vector constructsPCR efficiency (E) R² of Or- Target Primers Calibration der genes (5′ to3′) curve slope Description 1 Actin F - SEQ ID E = 109.8%, Referencegene NO: 28 R² = 0.995, R - SEQ ID Slope = −3.107 NO: 29 2 FPGS F - SEQID E = 104.9%, Reference gene NO: 30 R² = 0.990, R - SEQ ID Slope =−3.209 NO: 31 3 ZmWAK-RLK1 F - SEQ ID E = 104.0%, WAK-RLK1 = NO: 32 R² =0.996, ZmWAK-RLK1 R - SEQ ID Slope = −3.229 herein. NO: 33 4 BX1 (BX:F - SEQ ID E = 100.1%, Benzoxazinoid benzoxazinless) NO: 34 R² = 0.994,pathway R - SEQ ID Slope = −3.319 NO: 35 5 BX2 F - SEQ ID E = 126.1%,Benzoxazinoid NO: 36 R² = 0.989, pathway R - SEQ ID Slope = −2.821 NO:37 6 BX3 F - SEQ ID E = 107.4%, Benzoxazinoid NO: 38 R² = 0.994, pathwayR - SEQ ID Slope = −3.156 NO: 39 7 BX4 F - SEQ ID E = 100.9%,Benzoxazinoid NO: 40 R² = 0.994, pathway R - SEQ ID Slope = −3.300 NO:41 8 Bx5 F - SEQ ID E = 109.7%, Benzoxazinoid NO: 42 R² = 0.992, pathwayR - SEQ ID Slope = −3.109 NO: 43 9 BX6 F - SEQ ID E = 107.6%,Benzoxazinoid NO: 44 R² = 0.961, pathway R - SEQ ID Slope = −3.153 NO:45 10 BX7 F - SEQ ID E = 106.9%, Benzoxazinoid NO: 46 R² = 0.989,pathway R - SEQ ID Slope = −3.167 NO: 47 11 BX8 F - SEQ ID E = 114.0%,Benzoxazinoid NO: 48 R² = 0.987, pathway R - SEQ ID Slope = −3.027 NO:49 12 BX9 F - SEQ ID E = 117.3%, Benzoxazinoid NO: 50 R² = 0.999,pathway R - SEQ ID Slope = −2.967 NO: 51 13 BX10 + BX11 F - SEQ ID E =109.5%, Benzoxazinoid NO: 52 R² = 0.999, pathway R - SEQ ID Slope =−3.114 NO: 53 14 BX12 F - SEQ ID E = 95.4%, Benzoxazinoid NO: 54 R² =0.996, pathway R - SEQ ID Slope = −3.437 NO: 55 15 BX13 F - SEQ ID E =99.3%, Benzoxazinoid NO: 56 R² = 0.961, pathway R - SEQ ID Slope =−3.340 NO: 57 16 IGL (Indole F - SEQ ID E = 111.8%, BenzoxazinoidGlycerol NO: 58 R² = 0.996, pathway Phosphate R - SEQ ID Slope = −3.069Lyase) NO: 59 17 GLU1 (GLU: F - SEQ ID E = 110.6%, Benzoxazinoid betaNO: 60 R² = 0.998, pathway glucosidase) R - SEQ ID Slope = −3.092 NO: 6118 GLU2 F - SEQ ID E = 110.4%, Benzoxazinoid NO: 62 R² = 0.985, pathwayR - SEQ ID Slope = −3.095 NO: 63 19 ZmWAK-RLK1 F - SEQ ID plasmid NO: 64construction R - SEQ ID NO: 65

Example 4: Mycelium Development

The second leaves of 21-day seedling plants were harvested and cut into2×2 cm² leaf segments, which were placed and incubated on the phytoagarplates. A spore suspension (4.5×10⁴ spores/ml) was painted using swabson the leaf surface. The petri dishes carrying samples were sealed usingPARAFILM and incubated 24 hours at room temperature until harvest.Trypan blue straining was conducted as previously described (Chung,Chia-Lin, et al. “Resistance loci affecting distinct stages of fungalpathogenesis: use of introgression lines for QTL mapping andcharacterization in the maize-Setosphaeria turcica pathosystem.” BMCplant biology 10.1 (2010): 103). The infected segments at 1 dpi wereincubated overnight in an acetic acid:ethanol (1:3, v/v) solution, andthen in a mixed solution of acetic acid:ethanol:glycerol (1:5:1, v/v/v)for 4 hours. The samples were stained overnight in 0.01% (w/v) trypanblue lactophenol solution, and then washed once using ddH₂O and storedin 60% glycerol ready for use. Specimens were placed on slides andexamined under the ZEISS Axio Imager 2 microscope system (CARL ZEISS,Jena, Germany). The numbers of germinated spores, germ tubes,appressoria and successful penetrations (hyphae inside of cell orbetween cell walls) were counted. Three independent experiments wereperformed.

Example 5: RNA Extraction, RNA Sequencing and Data Analysis

The second leaves of seedling plants were harvested with four biologicalreplicates at 0, 9-hpi, 3-dpi and 10-dpi, which corresponded to beforeinoculation, the germination/penetration, biotrophic growth andnecrotrophic growth, respectively (Jennings, P. R., and A. J. Ullstrup.“A HISTOLOGICAL STUDY OF 3 HELMINTHOSPORIUM LEAF BLIGHTS OF CORN.”Phytopathology 47.12 (1957): 707-714; Hilu, H. M., and A. L. Hooker.“Host-pathogen relationship of Helminthosporium turcicum in resistantand susceptible corn seedlings.” (1964): 570-5). Forty-eight samples (4genotypes, 4 time points, 3 biological replicates) were subjected fortotal RNA extraction using SV Total RNA Isolation Kits (Promega,Dübendorf, Switzerland). 1 μl of total RNA was checked by Nanodrop 1000Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA) toestimate the RNA concentration. Meanwhile, 15 plants in each genotypewere evaluated for the AUDPC value to control if the infection worked.

The quantity and quality in RNA sequencing were determined using Qubit®1.0 Fluorometer (Thermo Fisher Scientific, Wilmington, USA) andBioanalyzer 2100 (Agilent, Waldbronn, Germany). The TruSeq Stranded mRNASample Prep Kit (Illumina, Inc., Hayward, USA) was used for librarypreparation. 1 μg of total RNA per sample was ribosome depleted and thensubjected for synthesizing double-strand cDNA. Each cDNA sample wasfragmented, end-repaired, polyadenylated and then ligated with TruSeqadaptor that contains the index for multiplexing. The cDNA fragmentscontaining TruSeq adapters at the both ends were enriched with PCRreaction. The enriched libraries were quantified and qualified, and thennormalized to 10 nM. The TruSeq SR Cluster Kit v4 cBot (Illumina, Inc.,Hayward, USA) was used for cluster generation using 8 pM of poolednormalized libraries. Sequencing was performed on the Illumina HiSeq2500at single end 125 bp using the TruSeq SBS Kit v4 (Illumina, Inc.,Hayward, USA).

The maize reference genome Zea_mays.AGPv3.27 and the correspondingannotation were downloaded (http://www.maizegdb.org/). The RNAsequencing reads were mapped on the reference genome with STAR (Dobin,Alexander, et al. “STAR: ultrafast universal RNA-seq aligner.”Bioinformatics 29.1 (2013): 15-21) allowing one mismatch per 100 bp andno multimapper with the following command: STAR—outFilterMultiMapNmax1—outFilterMismatchNoverLmax 0.01-alignIntronMax 10000. Read counts weredetermined from the mapping files with featureCounts 1.4.6 (Liao, Yang,Gordon K. Smyth, and Wei Shi. “featureCounts: an efficient generalpurpose program for assigning sequence reads to genomic features.”Bioinformatics 30.7 (2013): 923-930). Statistical analyses were donewith the R package edgeR and genes were tested for differentialexpression with pairwise comparisons and tagwise estimation ofdispersion. A gene was considered to be expressed when at least 10 readswere mapped on it and a gene was considered to be differentiallyexpressed with log 2FC≥|2| and FDR<0.01. First, pairwise comparisonswere performed between Htn1 and no Htn1 plants for each genotype andeach time points separately. The results were then compared between timepoints and then between the two genotypes. The Gene Ontology analysisfor differentially expressed genes (DEGs) was conducted by using onlinesoftware agriGO (Du, Zhou, et al. “agriGO: a GO analysis toolkit for theagricultural community.” Nucleic acids research 38.suppl_2 (2010):W64-W70). The significant terms were colored if adjusted p≤0.05.

Example 6: RT-qPCR Assay

1 μg total RNA was subjected for first strand cDNA synthesis using theiScript Advanced cDNA kit (172-5038, Rio-Rad). 1:20 diluted cDNA wasapplied for quantifying expression using a Real-Time System C1000TMThermal cycler (96 or 384 wells, Bio-Rad). The expression of targets wasnormalized by the reference genes FPGS and Actin as described (Hurni etal. 2015). The primers for expression analysis are shown in Table 1above.

Example 7: Benzoxazinoids (BXDs) Extraction and Measurement

60-100 mg leaves (without veins) of the seedling plants were harvestedand freezing immediately in liquid nitrogen, grinded and added theextraction buffer (1 mg sample+10 μl extraction buffer). The sampleswere mixed thoroughly and centrifuged at 13,000 rpm under 4° C. Thesupernatant was transferred into new tube and centrifuged once moreunder same condition, to remove the possible leaf particles. Thesupernatant was collected being ready for BXDs measurement.

Benzoxazinoid contents were analyzed by an Acquity UPLC equipment(Waters) coupled to a UV detector and coupled to a mass spectrometer(Waters) (Meihls, Lisa N., et al. “Natural variation in maize aphidresistance is associated with 2, 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside methyltransferase activity.” The Plant CellOnline 25.6 (2013): 2341-2355). An Acquity BEH C18 column (Waters) wasused. The temperatures of the autosampler and column were 15° C. and 40°C., respectively. The mobile phase consisted of 99% water, 1%acetonitrile, and 0.1% Formic acid (A) and acetonitrile and 0.1% Formicacid (B). Flow rate was set to 0.4 ml min⁻¹ with 3% A and 97% B followedby column reconditioning. The injection volume was 5 μl. The extractedtrace at 275 nm was used for benzoxazinoids quantification. Thefollowing extracted ion chromatograms were used for quantification witha mass window of ±0.01 D: mass-to-charge ratio (m/z) for DIMBOA(retention time [RT] 5.62 min) and DIMBOA-Glc (RT 5.64 min), m/z forHDMBOA-Glc (RT 8.19 min), m/z for HMBOA-Glc (RT 5.34 min) andDIM₂BOA-Glc (RT 5.825 min). Benzoxazinoids absolute concentrations weredetermined by external calibration curves obtained from purifiedDIMBOA-Glc, DIMBOA and HDMBOA-Glc standards.

Example 8: ZmWAK-RLK1 Encodes a Plasma Membrane Localized Protein

To determine the subcellular localization of the ZmWAK-RLK1 protein, afusion construct consisting of a full-length coding sequence fused tothe sequence of an enhanced green fluorescence protein (eGFP) at the Cterminus was generated (cf. SEQ ID NO:9 for the nucleic acid plasmidconstruct). The ZmWAK-RLK1 fusion protein localized to the plasmamembrane before and after plasmolysis when transiently expressed inonion epidermal cells. Furthermore, infiltration into leaves ofNicotiana benthamiana confirmed the localization of ZmWAK-RLK1 to theplasma membrane two days after infiltration (cf. FIG. 12). These data,particularly confocal analysis of onion epidermal cells after transientexpression of ZmWAK-RKL1-eGFP and the positive control PIP2A-mCherrythat is known to localize to the plasma membrane demonstrate thatZmWAK-RLK1 is a plasma membrane protein.

Example 9: ZmWAK-RLK1 Reduced Fungal Penetrations

Spores of the hemibiotrophic fungus E. turcicum penetrate the maizeepidermis mostly between 6-18 hours after inoculation (hpi) (Jenningsand Ullstrup, 1957). To investigate if ZmWAK-RLK1 changes the outcome offungal penetration attempts, we investigated the infection process atone day post inoculation (dpi) using trypan blue staining (data notshown). The number of successful penetration events were evaluated inthree EMS-induced ZmWAK-RLK1 loss-of-function mutant lines (RLK1b, RLK1dand RLK1f) and their corresponding sister lines that were generated inthe near isogenic line (NIL) RP3Htn1 (Hurni et al. 2015). No significantdifference in the establishment of germ tubes and appressoria wasobserved in genotypes with/without ZmWAK-RLK1 (data not shown). Incontrast, the number of successful penetration events was significantlylower if ZmWAK-RLK1 was functional compared to loss-of-function mutantsas demonstrated in FIGS. 2 A and B. This indicates that ZmWAK-RLK1 leadsto a reduction of pathogen penetrations into host tissues.

Example 10: Transcriptome and Metabolism Analysis Identified Alterationsto the BXDs Biosynthesis Pathway in the Presence of ZmWAK-RLK1

To decipher the immune network specifically influenced by ZmWAK-RLK1, weperformed a transcriptome analysis by RNA sequencing in two pairs ofnear isogenic lines, w22 and W22Htn1 as well as B37 and B37Htn1. NCLBdevelopment was significantly reduced in the presence of ZmWAK-RLK1 inboth NILs (FIG. 3 A-C). Leaf samples were collected at 0 and 9 hpi(penetration stage) as well as 3 (biotrophic growth) and 10 dpi(necrotrophic growth) (Jennings and Ullstrup, 1957). Forty-eight sampleswere sequenced and 1.159 billion reads were obtained (Table 2). Morethan 820 million reads were uniquely mapped with an average of 17.08million reads per sample (70.7%) (Table 2). No obvious difference ofpercentage of mapped reads in genotypes with or without Htn1 wasobserved. A total of 15,345 genes were expressed and they were used forfurther analysis. By conducting a multidimensional scaling (MDS)analysis using expression normalized by edgeR, the biological replicatesfor the same genotype-timepoints combination were mostly groupedtogether, suggesting the repeatability of replicates (data not shown).More differentially expressed gene (DEGs) were detected in B37Htn1/B37compared to w22Htn1/w22 (FIG. 4). These genes differently expressed inboth NILs in at least one of timepoints. To identify DEGs associatedwith ZmWAK-RLK1 and to rule out genetic background effects, only genesthat were differentially expressed in both NIL pairs were considered.

TABLE 2 Statistics of RNA-seq reads sequenced and mapped PercentagePercentage Uniquely of uniquely of multi- Percentage of Sample mappedmapped mapped unmapped number Samples Raw reads reads^(a) reads (%)reads (%) reads (%) 1 0-w22-1 19,894,158 12,757,882 64.13 12.66 20.34 20-w22-2 25,718,662 18,612,570 72.37 6.63 20.18 3 0-w22-3 24,937,38317,948,211 71.97 6.41 20.85 4 0-w22Htn1-1 27,452,042 20,264,307 73.825.96 19.70 5 0-w22Htn1-2 19,815,200 14,439,891 72.87 6.07 20.44 60-w22Htn1-3 24,173,527 16,215,534 67.08 9.55 21.40 7 0-B37-1 22,180,88814,689,311 66.23 5.27 28.03 8 0-B37-2 24,474,895 18,273,286 74.66 5.9618.75 9 0-B37-3 27,774,311 20,221,020 72.80 6.73 19.74 10 0-B37Htn1-126,728,646 19,277,404 72.12 6.16 21.04 11 0-B37Htn1-2 24,336,73117,640,859 72.49 6.06 20.82 12 0-B37Htn1-3 22,625,055 16,499,617 72.936.15 20.30 13 9h-w22-1 25,343,283 17,460,901 68.90 7.81 22.49 149h-w22-2 26,384,078 18,489,206 70.08 7.16 22.03 15 9h-w22-3 21,846,92413,680,343 62.62 5.97 30.81 16 9h-w22Htn1-1 34,074,874 23,121,230 67.856.27 25.40 17 9h-w22Htn1-2 24,276,403 17,299,847 71.26 7.02 21.07 189h-w22Htn1-3 30,200,422 20,381,456 67.49 7.23 24.62 19 9h-B37-117,962,777 12,878,288 71.69 7.38 20.10 20 9h-B37-2 23,815,810 16,892,47670.93 7.12 21.22 21 9h-B37-3 24,188,988 17,604,433 72.78 7.15 19.38 229h-B37Htn1-1 25,195,971 17,304,100 68.68 7.80 22.65 23 9h-B37Htn1-223,902,398 16,173,755 67.67 7.27 24.40 24 9h-B37Htn1-3 24,731,48117,279,056 69.87 7.13 22.39 25 3d-w22-1 22,399,000 15,772,632 70.42 6.4422.49 26 3d-w22-2 26,175,191 18,816,409 71.89 6.43 21.06 27 3d-w22-323,253,678 16,758,123 72.07 6.45 20.82 28 3d-w22Htn1-1 23,878,63917,185,076 71.97 5.92 21.58 29 3d-w22Htn1-2 20,568,384 14,338,420 69.717.09 22.35 30 3d-w22Htn1-3 34,008,596 24,127,141 70.94 5.81 22.76 313d-B37-1 23,690,913 14,726,042 62.16 4.70 32.71 32 3d-B37-2 27,646,35019,934,380 72.10 5.89 21.42 33 3d-B37-3 21,114,803 16,208,484 76.76 5.6916.98 34 3d-B37Htn1-l 21,758,134 15,409,264 70.82 5.68 23.03 353d-B37Htn1-2 27,236,811 19,866,766 72.94 5.84 20.68 36 3d-B37Htn1-322,700,637 16,553,064 72.92 5.60 21.00 37 10d-w22-1 23,051,84216,566,330 71.87 6.16 21.24 38 10d-w22-2 28,611,557 20,463,896 71.526.01 21.77 39 10d-w22-3 12,829,435 7,480,564 58.31 4.82 36.38 4010d-w22Htn1-1 24,435,356 17,199,294 70.39 5.45 23.76 41 10d-w22Htn1-228,996,022 21,442,119 73.95 5.78 19.69 42 10d-w22Htn1-3 18,649,09013,429,238 72.01 5.78 21.71 43 10d-B37-1 13,275,463 9,658,508 72.75 5.4621.16 44 10d-B37-2 15,217,491 11,288,257 74.18 5.38 19.89 45 10d-B37-323,349,541 17,096,788 73.22 5.36 20.83 46 10d-B37Htn1-1 20,104,13714,270,397 70.98 4.94 23.79 47 10d-B37Htn1-2 38,203,107 27,216,898 71.245.40 22.82 48 10d-B37Htn1-3 26,094,601 18,820,165 72.12 5.61 21.68 Total1,159,283,685 820,033,238 70.74 NA^(b) NA^(b) Mean 24,151,743 17,084,02670.74 NA^(b) NA^(b) ^(a)Parameters for mapping: less than 1% mismatch, 1locus mapped, intron size is less than 10 kb; ^(b)NA = not analyzed.

Two-hundred and fifteen common DEGs were identified across all timepoints (FIG. 13). 132 and 83 genes were induced and repressed in theZmWAK-RLK1-containing NIL compared to the respective susceptible line(heat map and Venn diagram not shown). 108 DEGs were only found attimepoint 0 before inoculation, while 107 of DEGs were found only afterinfection. Twenty-nine DEGs were differently expressed at all timepoints including timepoint. An overrepresentation analysis using agriGOwas performed to identify enriched Gene Ontology (GO) terms associatedwith ZmWAK-RLK1. The functional groups were enriched for terms ofdefense response (e.g. GO:0009814) and metabolic/biosynthetic process(e.g. GO:0006725) (data not shown).

To further analyze if the presence of ZmWAK-RLK1 is associated with BXDsbiosynthesis (FIG. 5 A), the content of major BXDs in second leaves ofw22Htn1 and w22 before and after infection was quantified (FIG. 5 B toF). The content of four BXDs DIMBOA-Glc, DIMBOA, HMBOA-Glc andDIM₂BOA-Glc was significantly lower in w22Htn1 compared to w22 at alltimepoints (data not shown), which indicated a constitutive reduction onBXDs content when ZmWAK-RLK1 is present. Furthermore, via RT-qPCR thetranscriptional levels of ZmWAK-RLK1 and specifically the genes in theBXDs biosynthesis pathway before and after pathogen inoculation weredetermined. The expression of genes (A) ZmWAK-RLK1, (B) Bx1, (C) Igl,(D) Bx2, (E) Bx3, (F) Bx4, (G) Bx5, (H) Bx6, (I) Bx7, (J) Bx8, (K) Bx9,(L) Bx10/11, (M) Bx12, (N) Bx13, (0) Glu1 and (P) Glu2 at differenttimepoints before and after infection was measured and statistics wereconducted separately in w22 and B37 genetic background using Tukey's HSD(P=0.05) in four biological replicates.

The ZmWAK-RLK1 expression in NILs showed no significant difference (FIG.11 A). Overall, the transcriptional levels of BXD biosynthesis geneswere genotype-specific (FIG. 11 A to P)). For instance, BX1 and itsprotein homolog IGL can convert indole-3-glycerol phosphate into indole,which is the first step of BXD metabolism (Frey et al. 2000). Theircoding genes Bx1 and Igl showed opposite contributions of geneexpression in B37 and w22, but the combined expression of Bx1 and Iglwas consistently lower in genotypes with ZmWAK-RLK1 (FIGS. 11 B and C).

Example 11: Mutations in ZmWAK-RLK1 is Associated with the Reduction ofSecondary Metabolite DIM₂BOA-Glc

To further analyze the role of different BXD biosynthesis genes as wellas the metabolites of this pathway in NCLB resistance, the ZmWAK-RLK1mutants (RLK1b, d and f, SEQ ID NOs: 3 to 6 and G548R mutant of SEQ IDNO:2) and their sister lines were used (Hurni et al. 2015). Thetranscript levels of several BXD genes were quantified and the contentof major BXD compounds in mutants which lost the resistance caused byZmWAK-RLK1 (FIG. 6 A to D). In contrast to the NILs, there was nodifference in the DIMBOA-Glc, DIMBOA and HMBOA, HDMBOA-Glcconcentration. However, the content of DIM₂BOA-Glc was significantlylower when ZmWAK-RLK1 was intact (FIG. 6 A, and further data not shown).The reduced DIM₂BOA-Glc correlated with a reduction in Igl transcriptlevels, while no obvious difference in the transcriptional levels ofZmWAK-RLK1 and Bx1 was detected in ZmWAK-RLK1 (FIG. 6 B to D) Bx6, Bx7and Bx13 are key genes of the BXDs pathway to produce DIM₂BOA-Glc, andthese genes were slightly but not significantly upregulated in mutants(data not shown). This phenomenon can be explained by a feedbackregulation. Therefore, ZmWAK-RLK1 clearly seems to be associated withthe reduction of secondary metabolite DIM₂BOA-Glc, possibly by reducingthe expression levels of Bx1 and/or Igl.

Example 12: Mutations in BXDs Biosynthesis Genes Decreased NCLBSusceptibility

To further analyze the role of BXDs in NCLB resistance/susceptibility,mutants in the three BXD biosynthesis genes Bx1, Bx2 and Bx6 were testedupon inoculation with E. turcicum. These mutants showed strong reductionin several BXDs compounds, including DIM₂BOA-Glc (FIG. 7).Interestingly, all three mutants showed a strong reduction of NCLBsusceptibility at the seedling stage (FIGS. 8 A and B). This confirmed anegative correlation of BXDs content and NCLB disease resistance.Furthermore, the ZmWAK-RLK1 expression at 10 dpi was checked (FIG. 8 C).No significant difference was detected in the bx mutants if compared tothe wild-type. Considering the reduction of Bx1 and Igl co-expression inZmWAK-RLK1 genotypes (FIG. 6 A, FIGS. 5 B and C, FIG. 9 B), the datasuggest that the BXDs synthesis pathway likely acts downstream ofZmWAK-RLK1.

Therefore, ZmWAK-RLK1 underlying quantitative NCLB disease resistance isbased on a decrease of the biosynthesis of secondary metabolite BXDs,and DIM₂BOA-Glc served as a candidate susceptibility component forpromoting fungal infection (FIG. 8 D).

Example 13: Tissue-Specific Expression of WAK-RLK1

Further to the determination of the subcellular localization of aZmWAK-RLK1 protein (see Example 8 above), the tissue specific expressionof RLK1 in different genotypes, and different time points wasdetermined. The genes FPGS and ACTIN served as reference to normalizethe expression. The results shown in FIG. 10 demonstrate that RLK1 is aubiquitously and steadily expressed gene which further underpins thefunction of said kinase as master regulator in relevant plant metabolicpathways.

Example 14: Tilling

To develop and screen a mutant population of plant material, TILLING wasperformed. A TILLING mutant population can be created, e.g., startingfrom KWS line RP3Htn1 according to Kato (2000, The maize handbook, pp.212-219)). Pollen is harvested from field-grown RP3Htn1 plants andtreated with 0.1% EMS solution for 45 min. Silks of individual plantsare then pollinated and emerging ears bagged. From 436 pollinated MOplants seeds were harvested, in one experiment. An additionalpropagation and selfing led to 10,084 individual M1 plants. Leafmaterial from these M1 plants was collected for DNA isolation. DNA ofdried leaf samples (10 leaf discs bunches/sample) was isolated from10,000 M1 individuals with the CTAB extraction method (Traitgenetics,Gatersleben, Germany). DNA is then aliquoted to 100 μl with 20 ng/μl.Primer development for mutant screening is performed. The amplificationassay consisted of 20 ng/μl DNA, 5×GoTaq-Buffer, 25 μM dNTPs, 10 μMforward Primer, 10 μM reverse Primer, 5 Units/μl GoTaq. Afterdenaturation for 300 s at 94° C. the amplification cycles were performedwith 35 cycles of 60 s at 94° C., 60 s at 60° C. and 60 s at 72° C.followed by a final elongation time for 600 s at 72° C. Next,Sanger-sequencing of PCR products is performed according to establishedprotocols. Sequences are then assembled, for example with the help ofthe software Lasergene Seqman NGen (DNASTAR) and heterozygote SNPscalled with the software default settings. Positive mutant plants weresequenced again with the Sanger-method in order to confirm thepolymorphism.

1.-15. (canceled)
 16. A method for producing a plant having increasedfungal resistance, wherein the fungal resistance is regulated by atleast one wall-associated kinase, the method comprising: (i) (a)providing at least one plant cell, tissue, organ, or whole plant havinga genotype with respect to the presence of at least one gene encoding awall-associated kinase in the genome of said plant cell, tissue, organ,or whole plant; or (i) (b) introducing at least one gene encoding atleast one wall-associated kinase into the genome of at least one cell ofat least one of a plant cell, tissue, organ, or whole plant; and (ii)(a) modifying at least one gene encoding at least one wall-associatedkinase in the at least one plant cell, tissue, organ, or whole plant;and/or (ii) (b) modulating the expression level of at least onewall-associated kinase and/or the transcription level, the expressionlevel, or a function of at least one molecule within a signallingpathway from the at least one wall-associated kinase to the synthesis ofat least one benzoxazinoid or within a synthesis pathway of at least onebenzoxazinoid in the at least one plant cell, tissue, organ, or wholeplant; (iii) producing a population of plants from the at least oneplant cell, tissue, organ, or whole plant; and (iv) selecting a planthaving increased fungal resistance from the population of plants,optionally based on a determination of a reduced synthesis of at leastone benzoxazinoid in response to a fungal pathogen infection, whereinthe synthesis of the at least one benzoxazinoid is regulated by the atleast one wall-associated kinase.
 17. The method according to claim 16,wherein the at least one wall-associated kinase is a WAK-RLK1 gene. 18.The method of claim 17, wherein the at least one wall-associated kinasea) is encoded by a nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO: 1 or 7, or a functional fragment thereof, b) isencoded by a nucleic acid molecule comprising the nucleotide sequencehaving at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to nucleotide sequence of SEQ ID NO: 1 or 7, c) isencoded a nucleic acid molecule hybridizing with a complementarysequence to a) or b) under stringent conditions, d) is encoded by anucleic acid molecule comprising the nucleotide sequence coding for anamino acid sequence of SEQ ID NO: 2 or 8, or a functional fragmentthereof, e) is encoded by a nucleic acid molecule comprising thenucleotide sequence coding for an amino acid sequence having at least60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity toamino acid sequence of SEQ ID NO: 2 or 8, f) comprising the amino acidsequence of SEQ ID NO: 2 or 8, or g) comprising an amino acid sequencehaving at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to amino acid sequence of SEQ ID NO: 2 or 8, providedthat any sequence of a) to g), optionally after expression, stillencodes at least one functional Htn1, Ht2, Ht3, or an allelic variant, amutant, or a functional fragment thereof.
 19. The method according toclaim 16, wherein the benzoxazinoid whose synthesis is regulated by theat least one wall-associated kinase, is selected from at least one ofDIMBOA, DIMBOA, HMBOA, HM2BOA, HDMBOA, HDM2BOA, HBOA, DHBOA, DIBOA orTRIBOA, the aforementioned benzoxazinoid being in the glucoside oraglucone form, or a benzoxazolinone, or any combination of theaforementioned benzoxazinoids.
 20. The method according to claim 16,wherein the fungal resistance against which resistance to a fungus isincreased, or a disease caused by said fungus is selected from a fungusof the order of Pleosporales, comprising Helminthosporium turcicumcausing northern corn leaf blight (NCLB), particularly affecting maizeand wheat plants, or comprising Bipolaris maydis causing southern cornleaf blight, the order of Pucciniales causing rust disease, comprisingPuccinia sorghi causing common rust, or Diploida macrospora causingDiploida leaf streak/blight, or Colletotrichum graminicola causingAnthracnose, or Fusarium spp. causing Fusarium stalk rot, or Gibberellaspp., causing Giberella stalk rot, or Sphacelotheca reiliana causingmaize head smut.
 21. The method according to claim 16, wherein the atleast one gene encoding at least one wall-associated kinase is stablyintegrated into the genome of the at least one plant cell, tissue,organ, or whole plant, or wherein the at least one gene encoding atleast one wall-associated kinase is transiently introduced into a plantcell, tissue, organ, or whole plant, or wherein the at least one geneencoding at least one wall-associated kinase is stably integrated intothe genome of the at least one plant cell, tissue, organ, or wholeplant, wherein the introduction of the at least one gene encoding atleast one wall-associated kinase comprises the introgression of the atleast one gene during plant breeding.
 22. The method according to claim16, wherein the at least one molecule within the signalling pathway fromthe at least one wall-associated kinase to the synthesis of at least onebenzoxazinoid or within the synthesis pathway of at least onebenzoxazinoid is selected from the group consisting of the genes bx1(SEQ ID NO: 10), bx2 (SEQ ID NO: 12), igl (SEQ ID NO: 14), bx6 (SEQ IDNO: 16), bxl1 (SEQ ID NO: 18), bxl4 (SEQ ID NO: 20), opr2 (SEQ ID NO:22), lox3 (SEQ ID NO: 24) or aoc1 (SEQ ID NO: 26), or a homologous genesthereof, or the proteins BX1 (SEQ ID NO: 11), BX2 (SEQ ID NO: 13), IGL(SEQ ID NO: 15), BX6 (SEQ ID NO: 17), BX11 (SEQ ID NO: 19), BX14 (SEQ IDNO: 21), OPR2 (SEQ ID NO: 23), LOX3 (SEQ ID NO: 25) or AOC1 gene (SEQ IDNO: 27), or a homolog thereof.
 23. The method according to claim 16,wherein the reduced synthesis of at least one benzoxazinoid is achievedby providing at least one wall-associated kinase, an allelic variant, amutant or a functional fragment thereof, or a gene encoding the same,wherein the at least one wall-associated kinase comprises a sequencewhich can directly or indirectly influence the benzoxazinoid pathway andat least one further plant metabolic pathway, wherein the plantmetabolic pathway is selected from the group consisting of the jasmonicacid pathway, the ethylene pathway, the lignin synthesis pathway, adefense pathway, a receptor-like kinase pathway, and a cell wallassociated pathway.
 24. The method according to claim 16, wherein themodification of the at least one gene encoding at least onewall-associated kinase within step (ii) (a) or (ii) (b) is performed byat least one of a site-specific nuclease (SSN) or a catalytically activefragment thereof, or a nucleic acid sequence encoding the same;oligonucleotide directed mutagenesis; chemical mutagenesis; or TILLING.25. The method according to claim 16, wherein the at least one plantcell, tissue, organ, or whole plant provided in step (i) is selectedfrom the group consisting of Hordeum vulgare, Hordeum bulbusom, Sorghumbicolor, Saccharum officinarium, Zea spp., including Zea mays, Setariaitalica, Oryza minuta, Oryza saliva, Oryza australiensis, Oryza alta,Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malusdomestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii,Daucus glochidiatus, Beta spp., including Beta vulgaris, Daucuspusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotianasylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotianabenthamiana, Solanum lycopersicum, Solanum tuberosum, Coffea canephora,Vitis vinifera, Erythrante guttata, Genlisea aurea, Cucumis sativus,Marus notabilis, Arabidopsis arenosa, Arabidopsis lyrata, Arabidopsisthaliana, Crucihimalaya himalaica, Crucihimalaya wallichii, Cardaminenexuosa, Lepidium virginicum, Capsella bursa pastoris, Olmarabidopsispumila, Arabis hirsute, Brassica napus, Brassica oleracea, Brassicarapa, Raphanus sativus, Brassica juncacea, Brassica nigra, Erucavesicaria subsp. sativa, Citrus sinensis, Jatropha curcas, Populustrichocarpa, Medicago truncatula, Cicer yamashitae, Cicer bijugum, Cicerarietinum, Cicer reticulatum, Cicer judaicum, Cajanus cajanifolius,Cajanus scarabaeoides, Phaseolus vulgaris, Glycine max, Gossypium sp.,Astragalus sinicus, Lotus japonicas, Torenia fournieri, Allium cepa,Allium fistulosum, Allium sativum, Helianthus annuus, Helianthustuberosus and Allium tuberosum, or any variety or subspecies belongingto one of the aforementioned plants.
 26. A plant cell, tissue, organ,whole plant or plant material, or a derivative or a progeny thereof,obtainable by a method according to claim
 16. 27. A method foridentifying at least one gene involved in increased pathogen resistancein a plant cell, tissue, organ, whole plant, or plant material themethod comprising: (i) determining the genotype of at least one plantcell, tissue, organ, whole plant, or plant material with respect to thepresence of at least one gene encoding a wall-associated kinase in thegenome of said plant cell, tissue, organ, whole plant or plant material;(ii) optionally determining the benzoxazinoid signature of the at leastone plant cell, tissue, organ, whole plant, or plant material of step(i); (iii) exposing the at least one plant cell, tissue, organ, wholeplant, or plant material of step (i) or (ii) to a stimulus, optionallywherein the stimulus is correlated with the benzoxazinoid signature inthe at least one plant cell, tissue, organ, whole plant, or plantmaterial; (iv) performing an analysis of at least one analyte obtainedfrom the at least one plant cell, tissue, organ, whole plant, or plantmaterial of step (i) or (ii) after exposition to the stimulus; (v)determining at least one gene being regulated upon exposition to astimulus according to step (iii) in at least one cell of the at leastone plant cell, tissue, organ, whole plant, or plant material asderivable from the analysis of at least one analyte as defined in step(iv), (vi) subjecting the at least one gene as determined in step (v) toa functional characterization; and (vii) providing at least one geneinvolved in increased pathogen resistance in a plant cell, tissue,organ, whole plant, or plant material.
 28. A plant cell, tissue, organ,whole plant or plant material, or a derivative or a progeny thereof,obtainable by introducing at least one gene as provided by the methodaccording to claim 27 into at least one cell of at least one of a plantcell, tissue, organ, or whole plant.
 29. A plant cell, tissue, organ,whole plant or plant material, or a derivative or a progeny thereof,wherein the introduction of at least one gene into at least one cell ofat least one of a plant cell, tissue, organ, or whole plant according tothe method according to claim 27 is a stable introduction.
 30. A methodof increasing pathogen resistance in a plant cell, tissue, organ, wholeplant, or plant material, the method comprising: (i) providing at leastone plant cell, tissue, organ, whole plant or plant material; (ii) (a)treating the at least one plant cell, tissue, organ, whole plant orplant material according to step (i) with a substance neutralizing aneffect of at least one benzoxazinoid, and/or (ii) (b) treating the atleast one plant cell, tissue, organ, whole plant or plant materialaccording to step (i) with a substance activating the signalling pathwaydownstream of at least one wall-associated kinase; and/or (ii) (c)treating the at least one plant cell, tissue, organ, whole plant orplant material according to step (i) with a substance modulating ormodifying the activity of at least one promoter or at least oneregulatory sequence of at least one gene of the at least one plant cell,tissue, organ, whole plant or plant material of step (i), wherein saidpromoter or said regulatory sequence is involved in the regulation oftranscription of at least on gene involved in the signalling pathway of,or downstream of at least one wall-associated kinase or involved in thesynthesis pathway of at least one benzoxazinoid; (ii) (d) treating theat least one plant cell, tissue, organ, whole plant or plant materialaccording to step (i) with a substance inhibiting the synthesis of atleast one benzoxazinoid; and (iii) reducing an amount of at least onebenzoxazinoid and thereby increasing pathogen resistance in at least oneplant cell, tissue, organ, whole plant, or plant material.
 31. Themethod of claim 30, wherein the method is effective to increase fungalresistance in at least one plant cell, tissue, organ, whole plant, orplant material.